Compositions and methods for managing or improving bone disorders, cartilage disorders, or both

ABSTRACT

The present disclosure provides mixtures of prenylated flavonoids, stilbenes, or both with flavans or curcuminoids or both capable of useful for promoting, managing or improving bone health, cartilage health or both, or for preventing or treating a bone disorder, cartilage disorder or both. Such a mixture of prenylated flavonoids, stilbenes, or both with flavans or curcuminoids or both can optionally be used in combination with other bone and cartilage management agents, such as calcium, magnesium, zinc, boron, vitamin D, vitamin K, glucosamine and/or chondroitin compounds, non-steroidal anti-inflammatory agents/analgesics, COX/LOX inhibiting agents, neuropathic pain relief agents, or the like.

BACKGROUND

Reduced bone density, which often evolves into osteoporosis in agingpopulations, especially pre- and post-menopausal women, is an importanthealth concern. Osteoporosis is defined clinically through themeasurement of bone mineral density (BMD), which remains the bestpredictor of primary osteoporotic fractures (Kanis et al., Osteoporos.Int. 16:581, 2005). Systemic inflammation is frequently associated withaccelerated bone reabsorption, which leads to bone loss. Variousmechanisms, such as elevated PGE2, TNF-α, IL-1β and otherpro-inflammatory cytokines, have been proposed to be involved with boneloss under inflammatory conditions (Hardy and Cooper, J. Endocrinol.201:309, 2009).

It has been shown that NSAIDs inhibit the COX enzyme and decreaseproduction of prostaglandins, which are involved in the regulation ofbone turnover (Raisz et al., Osteoporos. Int. 3(Suppl 1):136, 1993). Theuse of cyclooxygenase-2 (COX-2) inhibitors has been demonstrated notonly to impair load-induced bone formation, but also to preventmenopause-associated BMD loss (Richards et al., Osteoporos. Int.17:1410, 2006). For example, diclofenac is an NSAID that inhibits bothCOX-1 and COX-2 enzymes (Richards et al., 2006). In a human clinicaltrial, diclofenac was almost as effective as conjugated estrogens forprotection of bone loss in postmenopausal women (Bruce et al., Am. J.Med. 96:349, 1994). Cottrell et al. (Bone Joint Res. 2:41, 2013), havereported that a 5-lipoxygenase (LOX) inhibitor can enhance boneregeneration in an animal model. In human clinical trials inpostmenopausal women, regular use of the combination of a COX-2selective NSAID and aspirin has been shown to result in higher BMD atall skeletal sites, including whole body and total hip, as measured bybone density scanning (DXA) and both trabecular and cortical BMD of thelumbar spine by quantitative computer tomography (QCT) (Carbone et al.,J. Bone Miner. Res. 18:1795, 2003).

BRIEF SUMMARY

In brief, the present disclosure is directed to compounds andcompositions useful for management of bone disorders, cartilagedisorders or both, and to methods of improving bone health, cartilagehealth or both.

In certain embodiments, this disclosure provides a compositioncomprising a mixture of a Morus extract, optionally enriched for one ormore prenylated flavonoids (e.g., Diels-Alder adducts of a chalcone anda prenylphenyl moiety), or one or more stilbenes, or a combinationthereof, and an Acacia extract, optionally enriched for flavans. Infurther embodiments, this disclosure provides a composition comprising amixture of a Morus extract, optionally enriched for prenylatedflavonoids (e.g., Diels-Alder adducts of a chalcone and a prenylphenylmoiety), or one or more stilbenes, or a combination thereof, and anUncaria gambir extract, optionally enriched for flavans. In furtherembodiments, this disclosure provides a composition comprising a mixtureof a Morus extract enriched for one or more prenylated flavonoids (e.g.,Diels-Alder adducts of a chalcone and a prenylphenyl moiety), or one ormore stilbenes, or a combination thereof, and a Curcuma extract,optionally enriched for curcuminoids. In other embodiments, thisdisclosure provides a composition comprising a mixture of a Morusextract enriched for one or more prenylated flavonoids (e.g.,Diels-Alder adducts of a chalcone and a prenylphenyl moiety), or one ormore stilbenes, and a Peppermint extract. In other embodiments, any ofthe compositions further, optionally, contain one or more of calcium,vitamin D, glucosamine compounds, such as N-acetyl glucosamine, andother bioactive compounds.

For example, a mixture of Curcuma and Morus alba root-bark extracts in a1:1 ratio demonstrated beneficial synergistic effects with enhanced boneand cartilage health compared with either Curcuma or Morus albaroot-bark extracts alone.

In another aspect, the present disclosure provides methods for managingbone disorders, cartilage disorders, or both. In certain embodiments,the compositions of this disclosure can be used in methods for treating,preventing, or managing bone disorders, cartilage disorders, or both,minimizing bone reabsorption, reducing cartilage degradation, promotinghealthy bone density, protecting bone integrity, cartilage integrity orboth, diminishing the action of enzymes that affect bone health,cartilage health or both, increase or maintain bone density, improvingbone function, cartilage function or both, alleviating joint pain,alleviating joint stiffness, improving joint range of motion,flexibility or both, promoting mobility, or any combination thereof.

These and other aspects of the invention will be apparent upon referenceto the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows bone histomorphometry evaluated on both femur and tibia perknee joint by Micro CT scan in rats having MIA-induced osteoarthritis,with or without treatment with a composition comprising an Uncariaextract and a Morus extract.

DETAILED DESCRIPTION

In certain aspects, the present disclosure provides prenylatedflavonoids and resveratrol compounds mixed with flavans or curcuminoidsfor use in improving bone health, cartilage health or both. In certainembodiments, prenylated flavonoids and resveratrol compounds areextracted from Morus alba plant material, such as from the Morus albaroot. In other embodiments, a Morus extract combined with flavans isoptionally further combined with management agents for bone health,cartilage health or both, such as calcium (for example, found in theform of calcium citrate, calcium fructoborate, calcium carbonate,calcium lactate, calcium gluconate, or calcium phosphate), magnesium,boron, zinc, vitamin D, vitamin K, or other minerals and vitamins. Otherdietary supplements that promote joint health, such as glucosaminecompounds (like glucosamine sulfate, glucosamine hydrochloride, Nacetylglucosamine), chondroitin sulfate and methylsulfonylmethane,hyaluronic acid, ω-3 fatty acids (such as eicosapentaenoic acid, EPA anddocosahexaenoic acid, DHA), hydrolyzed collagen (e.g., from bovine typeI collagen, chicken sternal type II collagen), collagen derived peptidesor a mixture of collagen amino acids, xanthophyll carotenoids (e.g.,astaxanthin, which is distributed in marine bacteria, algae,crustaceans, fish); non-steroidal anti-inflammatory agents/analgesics,COX/LOX inhibitory agents (such as acetaminophen, ibuprofen, celecoxib);neuropathic pain relief agents, herbal or plant extracts (such as aBoswellia extract).

Other aspects relate to methods of using compositions of thisdisclosure, such as for maintaining bone structure, cartilage structureor both, minimizing bone reabsorption, preventing cartilage degradation,increasing bone density, promoting healthy joints by protectingcartilage integrity, diminishing the action of enzymes that affect bonehealth, cartilage health, or both, improving joint movement or function,alleviating joint pain, alleviating joint discomfort, alleviating jointpain and discomfort, alleviating joint stiffness, improving joint rangeof motion or flexibility, promote mobility, or the like.

In the following description, certain specific details are set forth inorder to provide a thorough understanding of various embodiments of thisdisclosure. However, one skilled in the art will understand that theinvention may be practiced without these details.

In the present description, any concentration range, percentage range,ratio range, or integer range is to be understood to include the valueof any integer within the recited range and, when appropriate, fractionsthereof (such as one tenth and one hundredth of an integer), unlessotherwise indicated. Also, any number range recited herein relating toany physical feature, such as polymer subunits, size or thickness, areto be understood to include any integer within the recited range, unlessotherwise indicated. As used herein, the terms “about” and “consistingessentially of” mean±20% of the indicated range, value, or structure,unless otherwise indicated. It should be understood that the terms “a”and “an” as used herein refer to “one or more” of the enumeratedcomponents. The use of the alternative (e.g., “or”) should be understoodto mean either one, both, or any combination thereof of thealternatives. Unless the context requires otherwise, throughout thepresent specification and claims, the word “comprise” and variationsthereof, such as, “comprises” and “comprising,” as well as synonymousterms like “include” and “have” and variants thereof, are to beconstrued in an open, inclusive sense; that is, as “including, but notlimited to.”

Reference throughout this specification to “one embodiment” or “anembodiment” means that a particular feature, structure or characteristicdescribed in connection with the embodiment is included in at least oneembodiment of the present invention. Thus, the appearances of thephrases “in one embodiment” or “in an embodiment” in various placesthroughout this specification are not necessarily all referring to thesame embodiment. Furthermore, the particular features, structures, orcharacteristics may be combined in any suitable manner in one or moreembodiments.

“Amino” refers to the —NH₂ radical.

“Cyano” refers to the —CN radical.

“Hydroxy” or “hydroxyl” refers to the —OH radical.

“Imino” refers to the ═NH substituent.

“Nitro” refers to the —NO₂ radical.

“Oxo” refers to the ═O substituent.

“Thioxo” refers to the ═S substituent.

“Alkyl” refers to a straight or branched hydrocarbon chain radicalconsisting solely of carbon and hydrogen atoms, which is saturated orunsaturated (i.e., contains one or more double or triple bonds), havingfrom one to twelve carbon atoms (C₁-C₁₂ alkyl), or one to eight carbonatoms (C₁-C₈ alkyl) or one to six carbon atoms (C₁-C₆ alkyl), and whichis attached to the rest of the molecule by a single bond, e.g., methyl,ethyl, n-propyl, 1-methylethyl (iso-propyl), n-butyl, n-pentyl,1,1-dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, ethenyl,prop-1-enyl, but-1-enyl, pent-1-enyl, penta-1,4-dienyl, ethynyl,propynyl, butynyl, pentynyl, hexynyl, and the like. Unless statedotherwise specifically in the specification, an alkyl group may beoptionally substituted.

“Alkylene” or “alkylene chain” refers to a straight or branched divalenthydrocarbon chain linking the rest of the molecule to a radical group,consisting solely of carbon and hydrogen, which is saturated orunsaturated (i.e., contains one or more double or triple bonds), andhaving from one to twelve carbon atoms, e.g., methylene, ethylene,propylene, n-butylene, ethenylene, propenylene, n-butenylene,propynylene, n-butynylene, and the like. The alkylene chain is attachedto the rest of the molecule through a single or double bond and to theradical group through a single or double bond. The points of attachmentof the alkylene chain to the rest of the molecule and to the radicalgroup can be through one carbon or any two carbons within the chain.Unless stated otherwise specifically in the specification, an alkylenechain may be optionally substituted.

“Alkoxy” refers to a radical of the formula —OR_(a) where R_(a) is analkyl radical as defined above containing one to twelve carbon atoms.Unless stated otherwise specifically in the specification, an alkoxygroup may be optionally substituted.

“Alkylamino” refers to a radical of the formula —NHR_(a) or —NR_(a)R_(a)where each R_(a) is, independently, an alkyl radical as defined abovecontaining one to twelve carbon atoms. Unless stated otherwisespecifically in the specification, an alkylamino group may be optionallysubstituted.

“Thioalkyl” refers to a radical of the formula —SR_(a) where R_(a) is analkyl radical as defined above containing one to twelve carbon atoms.Unless stated otherwise specifically in the specification, a thioalkylgroup may be optionally substituted.

“Aryl” refers to a hydrocarbon ring system radical comprising hydrogen,6 to 18 carbon atoms and at least one aromatic ring. For purposes ofthis disclosure, the aryl radical may be a monocyclic, bicyclic,tricyclic or tetracyclic ring system, which may include fused or bridgedring systems. Aryl radicals include aryl radicals derived fromaceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene,benzene, chrysene, fluoranthene, fluorene, as-indacene, s-indacene,indane, indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene,and triphenylene. Unless stated otherwise specifically in thespecification, the term “aryl” or the prefix “ar-” (such as in“aralkyl”) is meant to include aryl radicals that are optionallysubstituted.

“Aralkyl” refers to a radical of the formula —R_(b)—R_(c) where R_(b) isan alkylene chain as defined above and R_(c) is one or more arylradicals as defined above, for example, benzyl, diphenylmethyl and thelike. Unless stated otherwise specifically in the specification, anaralkyl group may be optionally substituted.

“Cycloalkyl” or “carbocyclic ring” refers to a stable non-aromaticmonocyclic or polycyclic hydrocarbon radical consisting solely of carbonand hydrogen atoms, which may include fused or bridged ring systems,having from three to fifteen carbon atoms, or having from three to tencarbon atoms, and which is saturated or unsaturated and attached to therest of the molecule by a single bond. Monocyclic radicals include, forexample, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,and cyclooctyl. Polycyclic radicals include, for example, adamantyl,norbornyl, decalinyl, 7,7-dimethyl-bicyclo[2.2.1]heptanyl, and the like.Unless otherwise stated specifically in the specification, a cycloalkylgroup may be optionally substituted.

“Cycloalkylalkyl” refers to a radical of the formula —R_(b)R_(d) whereR_(b) is an alkylene chain as defined above and R_(d) is a cycloalkylradical as defined above. Unless stated otherwise specifically in thespecification, a cycloalkylalkyl group may be optionally substituted.

“Fused” refers to any ring structure described herein which is fused toan existing ring structure in the compounds of this disclosure. When thefused ring is a heterocyclyl ring or a heteroaryl ring, any carbon atomon the existing ring structure which becomes part of the fusedheterocyclyl ring or the fused heteroaryl ring may be replaced with anitrogen atom.

“Halo” or “halogen” refers to bromo, chloro, fluoro or iodo.

“Haloalkyl” refers to an alkyl radical, as defined above, that issubstituted by one or more halo radicals, as defined above, e.g.,trifluoromethyl, difluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl,1,2-difluoroethyl, 3-bromo-2-fluoropropyl, 1,2-dibromoethyl, and thelike. Unless stated otherwise specifically in the specification, ahaloalkyl group may be optionally substituted.

“Heterocyclyl” or “heterocyclic ring” refers to a stable 3- to18-membered non-aromatic ring radical which consists of two to twelvecarbon atoms and from one to six heteroatoms selected from the groupconsisting of nitrogen, oxygen and sulfur. Unless stated otherwisespecifically in the specification, the heterocyclyl radical may be amonocyclic, bicyclic, tricyclic or tetracyclic ring system, which mayinclude fused or bridged ring systems; and the nitrogen, carbon orsulfur atoms in the heterocyclyl radical may be optionally oxidized; thenitrogen atom may be optionally quaternized; and the heterocyclylradical may be partially or fully saturated. Examples of suchheterocyclyl radicals include dioxolanyl, thienyl[1,3]dithianyl,decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl,isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl,2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl,piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl,quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl,tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl,1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl. Unless statedotherwise specifically in the specification, a heterocyclyl group may beoptionally substituted.

“N-heterocyclyl” refers to a heterocyclyl radical as defined abovecontaining at least one nitrogen and where the point of attachment ofthe heterocyclyl radical to the rest of the molecule is through anitrogen atom in the heterocyclyl radical. Unless stated otherwisespecifically in the specification, a N-heterocyclyl group may beoptionally substituted.

“Heterocyclylalkyl” refers to a radical of the formula —R_(b)R_(c) whereR_(b) is an alkylene chain as defined above and R_(e) is a heterocyclylradical as defined above, and if the heterocyclyl is anitrogen-containing heterocyclyl, the heterocyclyl may be attached tothe alkyl radical at the nitrogen atom. Unless stated otherwisespecifically in the specification, a heterocyclylalkyl group may beoptionally substituted.

“Heteroaryl” refers to a 5- to 14-membered ring system radicalcomprising hydrogen atoms, one to thirteen carbon atoms, one to sixheteroatoms selected from the group consisting of nitrogen, oxygen andsulfur, and at least one aromatic ring. For purposes of this disclosure,the heteroaryl radical may be a monocyclic, bicyclic, tricyclic ortetracyclic ring system, which may include fused or bridged ringsystems; and the nitrogen, carbon or sulfur atoms in the heteroarylradical may be optionally oxidized; the nitrogen atom may be optionallyquaternized. Examples include azepinyl, acridinyl, benzimidazolyl,benzothiazolyl, benzindolyl, benzodioxolyl, benzofuranyl, benzooxazolyl,benzothiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl,1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl,benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl,benzofuranonyl, benzothienyl (benzothiophenyl), benzotriazolyl,benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, cinnolinyl,dibenzofuranyl, dibenzothiophenyl, furanyl, furanonyl, isothiazolyl,imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indolinyl,isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl, naphthyridinyl,oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 1-oxidopyridinyl,1-oxidopyrimidinyl, 1-oxidopyrazinyl, 1-oxidopyridazinyl,1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl,phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl, pyridinyl,pyrazinyl, pyrimidinyl, pyridazinyl, quinazolinyl, quinoxalinyl,quinolinyl, quinuclidinyl, isoquinolinyl, tetrahydroquinolinyl,thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, andthiophenyl (i.e. thienyl). Unless stated otherwise specifically in thespecification, a heteroaryl group may be optionally substituted.

“N-heteroaryl” refers to a heteroaryl radical as defined abovecontaining at least one nitrogen and where the point of attachment ofthe heteroaryl radical to the rest of the molecule is through a nitrogenatom in the heteroaryl radical. Unless stated otherwise specifically inthe specification, an N-heteroaryl group may be optionally substituted.

“Heteroarylalkyl” refers to a radical of the formula —R_(b)R_(f) whereR_(b) is an alkylene chain as defined above and R_(f) is a heteroarylradical as defined above. Unless stated otherwise specifically in thespecification, a heteroarylalkyl group may be optionally substituted.

The term “substituted” used herein means any of the above groups (i.e.,alkyl, alkylene, alkoxy, alkylamino, thioalkyl, aryl, aralkyl,cycloalkyl, cycloalkylalkyl, haloalkyl, heterocyclyl, N-heterocyclyl,heterocyclylalkyl, heteroaryl, N-heteroaryl or heteroarylalkyl), whereinat least one hydrogen atom is replaced by a bond to a non-hydrogen atomssuch as a halogen atom such as F, Cl, Br, and I; an oxygen atom ingroups such as hydroxyl groups, alkoxy groups, and ester groups; asulfur atom in groups such as thiol groups, thioalkyl groups, sulfonegroups, sulfonyl groups, and sulfoxide groups; a nitrogen atom in groupssuch as amines, amides, alkylamines, dialkylamines, arylamines,alkylarylamines, diarylamines, N-oxides, imides, and enamines; a siliconatom in groups such as trialkylsilyl groups, dialkylarylsilyl groups,alkyldiarylsilyl groups, and triarylsilyl groups; and other heteroatomsin various other groups. “Substituted” also means any of the abovegroups in which one or more hydrogen atoms are replaced by ahigher-order bond (e.g., a double- or triple-bond) to a heteroatom, suchas oxygen in oxo, carbonyl, carboxyl, and ester groups; and nitrogen ingroups such as imines, oximes, hydrazones, and nitriles. For example,“substituted” includes any of the above groups in which one or morehydrogen atoms are replaced with —NR_(g)R_(h), —NR_(g)C(═O)R_(h),—NR_(g)C(═O)NR_(g)R_(h), —NR_(g)C(═O)OR_(h), —NR_(g)SO₂R_(h),—OC(═O)NR_(g)R_(h), —OR_(g), —SR_(g), —SOR_(g), —SO₂R_(g), —OSO₂R_(g),—SO₂OR_(g), ═NSO₂R_(g), and —SO₂NR_(g)R_(h). “Substituted” also meansany of the above groups in which one or more hydrogen atoms are replacedwith —C(═O)R_(g), —C(═O)OR_(g), —C(═O)NR_(g)R_(h), —CH₂SO₂R_(g),—CH₂SO₂NR_(g)R_(h). In the foregoing, R_(g) and R_(h) are the same ordifferent and independently hydrogen, alkyl, alkoxy, alkylamino,thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, haloalkyl,heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl,N-heteroaryl or heteroarylalkyl. “Substituted” further means any of theabove groups in which one or more hydrogen atoms are replaced by a bondto an amino, cyano, hydroxyl, imino, nitro, oxo, thioxo, halo, alkyl,alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl,cycloalkylalkyl, haloalkyl, heterocyclyl, N-heterocyclyl,heterocyclylalkyl, heteroaryl, N-heteroaryl or heteroarylalkyl group. Inaddition, each of the foregoing substituents may also be optionallysubstituted with one or more of the above substituents.

“Glycoside” refers to a molecule in which a sugar group is bondedthrough its anomeric carbon to another group via a glycosidic bond.Exemplary sugars include glucose, rhamnose, manose, galactose,arabinose, glucuronide and others. Glycosides can be linked by an O- (anO-glycoside), N- (a glycosylamine), S- (a thioglycoside), or C- (aC-glycoside) glycosidic bond. Compounds of this disclosure can formglycosides at any suitable attachment point.

A “prenyl group” is a moiety comprising a five-carbon backbone of thefollowing structure:

In some embodiments, prenyl groups comprise one or more carbon-carbondouble bonds and/or are substituted with one or more substituents.“Prenyl” refers to the

radical. Isoprenyl refers to the

radical (cis or trans). Prenyl groups are substituted or unsubstituted,such as

“Prenylphenyl” refers to a phenyl moiety connected to a prenyl moiety asdefined above. Prenylphenyls include substituted phenyls such asflavonoids and other substituted phenyls and heteroaryls, provided thereis at least one prenyl group in the molecule. In the case of substitutedphenyls and heteroaryl, the prenyl moiety need not be directly attachedto the phenyl ring, but can be attached at any point in the molecule.

“Chalcone” refers to a compound comprising the following core structure:

Chalcones can be variously substituted at any of the above carbon atoms.

“Prodrug” is meant to indicate a compound that may be converted underphysiological conditions or by solvolysis to a biologically activecompound of this disclosure. Thus, the term “prodrug” refers to ametabolic precursor of a compound of this disclosure that ispharmaceutically and nutraceutically acceptable. A prodrug may beinactive when administered to a subject in need thereof, but isconverted in vivo to an active compound of this disclosure. Prodrugs aretypically rapidly transformed in vivo to yield the parent compound ofthis disclosure, for example, by hydrolysis in blood or intestine ormetabolized in the liver. The prodrug compound often offers advantagesof solubility, tissue compatibility or delayed release in a mammalianorganism (see Bundgard, H., Design of Prodrugs (1985), pp. 7-9, 21-24(Elsevier, Amsterdam)). A discussion of prodrugs is provided in Higuchi,T., et al., A.C.S. Symposium Series, Vol. 14, and in BioreversibleCarriers in Drug Design, Ed. Edward B. Roche, American Pharmaceuticaland Nutraceutical Association and Pergamon Press, 1987.

The term “prodrug” is also meant to include any covalently bondedcarriers, which release the active compound of this disclosure in vivowhen such prodrug is administered to a mammalian subject. Prodrugs of acompound of this disclosure may be prepared by modifying functionalgroups present in the compound of this disclosure in such a way that themodifications are cleaved, either in routine manipulation or in vivo, tothe parent compound of this disclosure. Prodrugs include compounds ofthis disclosure wherein a hydroxy, amino or mercapto group is bonded toany group that, when the prodrug of the compound of this disclosure isadministered to a mammalian subject, cleaves to form a free hydroxy,free amino or free mercapto group, respectively. Examples of prodrugsinclude acetate, formate and benzoate derivatives of alcohol or amidederivatives of amine functional groups in the compounds of thisdisclosure and the like.

The instant disclosure is also meant to encompass all pharmaceuticallyor nutraceutically acceptable compounds of any one of structures(I)-(VI) being isotopically-labelled by having one or more atomsreplaced by an atom having a different atomic mass or mass number.Examples of isotopes that can be incorporated into the disclosedcompounds include isotopes of hydrogen, carbon, nitrogen, oxygen,phosphorous, fluorine, chlorine, and iodine, such as ²H, ³H, ¹¹C, ¹³C,¹⁴C, ¹³N, ¹⁵N, ¹⁵O, ¹⁷O, ¹⁸O ³¹P, ³²P, ³⁵S, ¹⁸F, ³⁶Cl, ¹²³I, and ¹²⁵I,respectively. These radiolabelled compounds could be useful to helpdetermine or measure the effectiveness of the compounds, bycharacterizing, for example, the site or mode of action, or bindingaffinity to pharmacologically important site of action. Certainisotopically-labelled compounds of any one of structures (I)-(VI), forexample, those incorporating a radioactive isotope, are useful in drugor substrate tissue distribution studies. The radioactive isotopestritium, i.e., ³H, and carbon-14, i.e., ¹⁴C, are particularly useful forthis purpose in view of their ease of incorporation and ready means ofdetection.

Substitution with heavier isotopes such as deuterium, i.e., ²H, mayafford certain therapeutic advantages resulting from greater metabolicstability, for example, increased in vivo half-life or reduced dosagerequirements, and hence may be preferred in some circumstances.

Substitution with positron emitting isotopes, such as ¹¹C, ¹⁸F, ¹⁵O and¹³N, can be useful in Positron Emission Topography (PET) studies forexamining substrate receptor occupancy. Isotopically-labeled compoundsof any one of structures (I)-(VI) can generally be prepared byconventional techniques known to those skilled in the art or byprocesses analogous to those described in the preparations and examplesas set out herein using an appropriate isotopically-labeled reagent inplace of the non-labeled reagent previously employed.

The instant disclosure is also meant to encompass the in vivo metabolicproducts of the disclosed compounds. Such products may result from, forexample, the oxidation, reduction, hydrolysis, amidation,esterification, and the like of the administered compound, primarily dueto enzymatic processes. Accordingly, this disclosure includes compoundsproduced by a process comprising administering a compound of thisdisclosure to a mammal for a period of time sufficient to yield ametabolic product thereof. Such products are typically identified byadministering a radiolabelled compound of this disclosure in adetectable dose to an animal, such as rat, mouse, guinea pig, dog, cat,pig, sheep, horse, monkey, or human, allowing sufficient time formetabolism to occur, and isolating its conversion products from theurine, blood or other biological samples.

“Stable compound” and “stable structure” are meant to indicate acompound that is sufficiently robust to survive isolation to a usefuldegree of purity from a reaction mixture, and formulation into anefficacious therapeutic agent.

“Mammal” includes humans, domestic animals (such as laboratory animalsor household pets like rat, mouse, guinea pig, cats, dogs, swine,cattle, sheep, goats, horses, rabbits, primates), and non-domesticanimals (such as wildlife) or the like.

“Optional” or “optionally” means that the subsequently describedelement, component, event or circumstances may or may not occur, andincludes instances where the element, component, event or circumstanceoccur and instances in which they do not. For example, “optionallysubstituted aryl” means that the aryl radical may or may not besubstituted—in other words, the description includes both substitutedaryl radicals and aryl radicals having no substitution.

“Pharmaceutically or nutraceutically acceptable carrier, diluent orexcipient” includes any adjuvant, carrier, excipient, glidant,sweetening agent, diluent, preservative, dye/colorant, flavor enhancer,surfactant, wetting agent, dispersing agent, suspending agent,stabilizer, isotonic agent, solvent, or emulsifier which has beenapproved by the United States Food and Drug Administration as beingacceptable for use in humans or domestic animals.

“Pharmaceutically or nutraceutically acceptable salt” includes both acidand base addition salts.

“Pharmaceutically or nutraceutically acceptable acid addition salt”refers to those salts which retain the biological effectiveness andproperties of the free bases, which are not biologically or otherwiseundesirable, and which are formed with inorganic acids such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid and the like, and organic acids such as acetic acid,2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid,aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoicacid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproicacid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamicacid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonicacid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid,galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid,glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid,glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid,lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid,malonic acid, mandelic acid, methanesulfonic acid, mucic acid,naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid,1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid,oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamicacid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid,stearic acid, succinic acid, tartaric acid, thiocyanic acid,p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, or thelike.

“Pharmaceutically or nutraceutically acceptable base addition salt”refers to those salts which retain the biological effectiveness andproperties of the free acids, which are not biologically or otherwiseundesirable. These salts are prepared from addition of an inorganic baseor an organic base to the free acid. Salts derived from inorganic basesinclude the sodium, potassium, lithium, ammonium, calcium, magnesium,iron, zinc, copper, manganese, aluminum salts and the like. In certainembodiments, the inorganic salts are ammonium, sodium, potassium,calcium, or magnesium salts. Salts derived from organic bases includesalts of primary, secondary, and tertiary amines, substituted aminesincluding naturally occurring substituted amines, cyclic amines andbasic ion exchange resins, such as ammonia, isopropylamine,trimethylamine, diethylamine, triethylamine, tripropylamine,diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol,2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine,procaine, hydrabamine, choline, betaine, benethamine, benzathine,ethylenediamine, glucosamine, methylglucamine, theobromine,triethanolamine, tromethamine, purines, piperazine, piperidine,N-ethylpiperidine, polyamine resins and the like. Particularly usefulorganic bases include isopropylamine, diethylamine, ethanolamine,trimethylamine, dicyclohexylamine, choline, or caffeine.

Often crystallizations produce a solvate of the compound of thisdisclosure. As used herein, the term “solvate” refers to an aggregatethat comprises one or more molecules of a compound of this disclosurewith one or more molecules of solvent. The solvent may be water, inwhich case the solvate may be a hydrate. Alternatively, the solvent maybe an organic solvent. Thus, the compounds of the present disclosure mayexist as a hydrate, including a monohydrate, dihydrate, hemihydrate,sesquihydrate, trihydrate, tetrahydrate and the like, as well as thecorresponding solvated forms. The compound of this disclosure may betrue solvates, while in other cases, a compound of this disclosure maymerely retain adventitious water or be a mixture of water plus someadventitious solvent.

A “pharmaceutical composition” or “nutraceutical composition” refers toa formulation of a compound of this disclosure and a medium generallyaccepted in the art for the delivery of the biologically active compoundto mammals, e.g., humans. For example, a pharmaceutical composition ofthe present disclosure may be formulated or used as a stand alonecomposition, or as a component in a prescription drug, anover-the-counter (OTC) medicine, a botanical drug, an herbal medicine, ahomeopathic agent, functional foods, or any other form of health careproduct reviewed and approved by a government agency. Exemplarynutraceutical compositions of the present disclosure may be formulatedor used as a stand alone composition, or as a nutritional or bioactivecomponent in food, a novel food, a functional food, a beverage, a bar, afood flavor, a food additive, a medical food, a dietary supplement, oran herbal product. A medium generally accepted in the art includes allpharmaceutically or nutraceutically acceptable carriers, diluents orexcipients therefor.

As used herein, “enriched for” refers to a plant extract or otherpreparation having at least a two-fold up to about a 1000-fold increasein the amount or activity of one or more active compounds as compared tothe amount or activity of the one or more active compounds found in theweight of the plant material or other source before extraction or otherpreparation. In certain embodiments, the weight of the plant material orother source before extraction or other preparation may be dry weight,wet weight, or a combination thereof.

As used herein, “major active ingredient” or “major active component”refers to one or more active compounds found in a plant extract or otherpreparation, or enriched for in a plant extract or other preparation,which is capable of at least one biological activity. In certainembodiments, a major active ingredient of an enriched extract will bethe one or more active compounds that were enriched in that extract.Generally, one or more major active components will impart, directly orindirectly, most (i.e., greater than 50%) of one or more measurablebiological activities or effects as compared to other extractcomponents. In certain embodiments, a major active ingredient may be aminor component by weight percentage of an extract (e.g., less than 50%,25%, 20%, 15%, 10%, 5%, or 1% of the components contained in an extract)but still provide most of the desired biological activity. Anycomposition of this disclosure containing a major active ingredient mayalso contain minor active ingredients that may or may not contribute tothe pharmaceutical or nutraceutical activity of the enrichedcomposition, but not to the level of major active components, and minoractive components alone may not be effective in the absence of a majoractive ingredient.

“Effective amount” or “therapeutically effective amount” refers to thatamount of a compound or composition of this disclosure that, whenadministered to a mammal, such as a human, is sufficient to effecttreatment, including any one or more of: (1) treating or preventing lossof bone and cartilage in a mammal; (2) promoting bone and cartilagehealth; (3) suppressing loss of bone and cartilage in a mammal; (4)increasing bone density in a mammal; (5) treating or preventingeosteoporosis in a mammal; (6) modifying inflammation of bone andcartilage in a mammal; and (7) protecting bone and cartilage integrity.The amount of a compound or composition of this disclosure thatconstitutes a “therapeutically effective amount” will vary depending onthe compound, the condition being treated and its severity, the mannerof administration, the duration of treatment, or the body weight and ageof a subject to be treated, but can be determined by one of ordinaryskill in the art having regard to his own knowledge and to thisdisclosure.

“Supplements” as used herein refers to a product that improves,promotes, supports, increases, regulates, manages, controls, maintains,optimizes, modifies, reduces, inhibits, or prevents a particularcondition, structure or function associated with a natural state orbiological process (i.e., are not used to diagnose, treat, mitigate,cure, or prevent disease). In certain embodiments, a supplement is adietary supplement. For example, with regard to bone and cartilagehealth-related conditions, dietary supplements may be used to maintainbone and cartilage integrity, minimize bone reabsorption, minimizecartilage degradation, promote healthy bone and cartilage by protectingbone and cartilage integrity, diminish the action of enzymes that affectbone and cartilage health, improve oesteoprosis condition, support bonerebuild, alleviate pain, alleviate discomfort, alleviate stiffness,improve range of motion, improve flexibility, promote mobility, or thelike. In certain embodiments, dietary supplements are a special categoryof diet, food or both, and are not a drug.

“Treating” or “treatment” or “ameliorating” refers to either atherapeutic treatment or prophylactic/preventative treatment of adisease or condition of interest in a mammal, such as a human, having orsuspected of having a disease or condition of interest, and includes:(i) preventing the disease or condition from occurring in a mammal, inparticular, when such mammal is predisposed to the condition but has notyet been diagnosed as having it; (ii) inhibiting the disease orcondition, i.e., arresting its development; (iii) relieving the diseaseor condition, i.e., causing regression of the disease or condition; or(iv) relieving the symptoms resulting from the disease or condition,(e.g., relieving pain, reducing inflammation, reducing loss ofcartilege, increasing bone density) without addressing the underlyingdisease or condition. As used herein, the terms “disease” and“condition” may be used interchangeably or may be different in that theparticular malady or condition may not have a known causative agent (sothat etiology has not yet been worked out) and it is therefore not yetrecognized as a disease but only as an undesirable condition orsyndrome, wherein a more or less specific set of symptoms have beenidentified by clinicians. In certain embodiments, compositions andmethods of the instant disclosure are useful for treating, managing orameliorating, for example, osteoarthritis, rheumatoid arthritis, orboth.

As used herein, “statistical significance” refers to a p value of 0.050or less as calculated using the Students t-test and indicates that it isunlikely that a particular event or result being measured has arisen bychance.

The chemical naming protocol and structure diagrams used herein are amodified form of the I.U.P.A.C. nomenclature system, using the ACD/NameVersion 9.07 software program or ChemDraw Ultra Version 11.0 softwarenaming program (CambridgeSoft), wherein the compounds of this disclosureare named herein as derivatives of the central core structure, e.g., theimidazopyridine structure. For complex chemical names employed herein, asubstituent group is named before the group to which it attaches. Forexample, cyclopropylethyl comprises an ethyl backbone with cyclopropylsubstituent. Except as described below, all bonds are identified in thechemical structure diagrams herein, except for some carbon atoms, whichare assumed to be bonded to sufficient hydrogen atoms to complete thevalency.

As noted herein, in certain embodiments, the present disclosure providesa composition comprising prenylated flavonoids. Flavonoids includeflavans, flavones, flavonols, flavanones, flavanonols, isoflavonoids,neoflavonoids, chalcones, arylbenzofuran, or the like.

In certain embodiments, a flavonoid compound of the present disclosurehas structure (III), as follows:

wherein R₁-R₁₂ are each independently H, hydroxyl, a prenyl group,chalcone, glycoside, halogen, sulfhydryl, amino, aldehyde, C₁₋₁₂ alkyl,C₁₋₁₂ alkoxy, C₁₋₁₂ alkthio, C₁₋₁₂ alkyamino, cycloalkyl, heterocyclyl,aryl, heteroaryl, aralkyl, alkylcarbonyl, aralkylcarbonyl, or a bond toa compound of structure (III) or (IV); or one of R₁-R₁₂ joins withanother one of R₁-R₁₂ to form a ring, and the remaining R₁-R₁₂ are eachindependently H, hydroxyl, a prenyl group, flavonoid, chalcone,glycoside, halogen, sulfhydryl, amino, aldehyde, C₁₋₁₂ alkyl, C₁₋₁₂alkoxy, C₁₋₁₂ alkthio, C₁₋₁₂ alkyamino, cycloalkyl, heterocyclyl, aryl,heteroaryl, aralkyl, alkylcarbonyl, aralkylcarbonyl or a bond to acompound of structure (III) or (IV), provided that all valencies aresatisfied (e.g., when the optional double bond is present in ring C,then R₁₂ is absent and at least one of R₁₀ or R₁₁ is absent). In certainembodiments, at least one of R₁-R₁₂ is a prenyl group, such as

In further embodiments, the optional double bond is present in ring C,R₁₁ and R₁₂ are absent, and R₁₀ is a prenyl group. In still furtherembodiments, at least one of R₁-R₉ is a prenyl group and R₁₀-R₁₂ areindependently H or hydroxyl. In certain specific embodiments, theprenylated flavonoids include Albanin G, Kuwanon G, Morusin, or anycombination thereof.

In certain embodiments, a flavonoid compound of the present disclosurehas structure (IV) as follows:

wherein R₁-R₁₂ are each independently H, hydroxyl, a prenyl group,flavonoid, chalcone, glycoside, halogen, sulfhydryl, amino, aldehyde,C₁₋₁₂ alkyl, C₁₋₁₂ alkoxy, C₁₋₁₂ alkthio, C₁₋₁₂ alkyamino, cycloalkyl,heterocyclyl, aryl, heteroaryl, aralkyl, alkylcarbonyl, aralkylcarbonyl,or a bond to a compound of structure (III) or (IV); or one of R₁-R₁₂joins with another one of R₁-R₁₂ to form a ring, and the remainingR₁-R₁₂ are each independently H, hydroxyl, a prenyl group, flavonoid,chalcone, glycoside, halogen, sulfhydryl, amino, aldehyde, C₁₋₁₂ alkyl,C₁₋₁₂ alkoxy, C₁₋₁₂ alkthio, C₁₋₁₂ alkyamino, cycloalkyl, heterocyclyl,aryl, heteroaryl, aralkyl, alkylcarbonyl, aralkylcarbonyl or a bond to acompound of structure (III) or (IV), provided that all valencies aresatisfied (e.g., when the optional double bond is present in ring C,then R₁₂ is absent and at least one of R₁₀ or R₁₁ is absent). In certainembodiments, at least one of R₁-R₁₂ is a prenyl group, such as

In further embodiments, the optional double bond is present in ring C,R₁₁ and R₁₂ are absent, and R₁₀ is a prenyl group. In still furtherembodiments, at least one of R₁-R₉ is a prenyl group and R₁₀-R₁₂ areindependently H or hydroxyl. In certain specific embodiments, theprenylated flavonoids include Albanin G, Kuwanon G, Morusin, morusinol,Sanggenon, isoxanthoumol, glabridin, cathayanon A, or any combinationthereof.

In some embodiments, a chalconoid compound of the present disclosure hasstructure (V) as follows:

wherein R₁-R₁₀ are each independently H, hydroxyl, a prenyl group,flavonoid, chalcone, glycoside, halogen, sulfhydryl, amino, aldehyde,C₁₋₁₂ alkyl, C₁₋₁₂ alkoxy, C₁₋₁₂ alkthio, C₁₋₁₂ alkyamino, cycloalkyl,heterocyclyl, aryl, heteroaryl, aralkyl, alkylcarbonyl, oraralkylcarbonyl. In certain embodiments, at least one of R₁-R₁₀ is aprenyl group, such as

In further embodiments, the optional double bond is present in ring C,R₁₁ and R₁₂ are absent, and R₁₀ is a prenyl group. In still furtherembodiments, at least one of R₁-R₉ is a prenyl group and R₁₀-R₁₂ areindependently H or hydroxyl. In certain specific embodiments, achalconoid compound includes xanthohumol.

In certain embodiments, a stilbene compound of the present disclosure isan (E)-stilbene (trans isomer) structure of formula I or (Z)-stilbene(cis isomer) structure of formula II, as follows:

wherein R₁-R₁₀ are each independently H, hydroxyl, glycoside, a prenylgroup, flavonoid, chalcone, halogen, sulfhydryl, amino, aldehyde, C₁₋₁₂alkyl, C₁₋₁₂ alkenyl, C₁₋₁₂ alkoxy, C₁₋₁₂ alkthio, C₁₋₁₂ alkyamino,cycloalkyl, heterocyclyl, aryl, heteroaryl, aralkyl, alkyl carbonyl, oraralkylcarbonyl. In certain embodiments, at least one of R₁-R₁₂ is aprenyl group, such as

In further embodiments, R₁, R₅, R₆ and R₁₀ are H. In still furtherembodiments, R₂ is a glucoside, or R₂ and R₈ are glycosides, and one ormore of R₄, R₉, and R₁₀ are hydroxyl. In yet further embodiments, R₁,R₅, and R₆ are H, and one or more of R₂-R₄ and R₇-R₁₀ are independentlyhydroxyl, C₁₋₃ alkoxy, or any combination thereof. In certain specificembodiments, a stilbene includes oxyresveratrol, resveratrol,piceatannol, pinosylvin, 3,4′-dihydroxystilbene, combretastatin A-1,pterostilbene, rhapontigenin, and a stilbene glycoside includesmulberroside A, rhaponticin, piceid, astringin, or any combination ofthese stilbenes or stilbene glycosides.

It is understood that any embodiment of the compounds of structure (I)to (VI), as set forth above, and any specific substituent set forthherein for the compounds of structure (I) to (VI), may be independentlycombined with other embodiments or substituents of any one of thecompounds of structure (I) to (VI) to form embodiments of thisdisclosure not specifically set forth above. In addition, in the eventthat a list of substituents is listed for any particular R group in aparticular embodiment or claim, it is understood that each individualsubstituent may be deleted from the particular embodiment or claim andthat the remaining list of substituents will be considered to be withinthe scope of this disclosure.

For the purposes of administration, compounds and compositions of thepresent disclosure may be administered as a raw chemical or may beformulated as pharmaceutical or nutraceutical compositions. In certainembodiments, pharmaceutical or nutraceutical compositions of the presentdisclosure comprise any one or more of the compounds having structure(I) to (VI) and a pharmaceutically or nutraceutically acceptablecarrier, diluent or excipient. The compounds of structures (I) to (VI)are individually or in combination present in the composition in anamount that is effective to treat a particular disease or condition ofinterest. Promoting, managing, or improving joint health or treatingdisease with compounds as set forth in any one of structures (I) to (VI)can be determined by one skilled in the art, for example, as describedin the Examples herein.

In certain embodiments, compounds and compositions (e.g.,pharmaceutical, nutraceutical) of the present disclosure may beadministered in an amount sufficient to promote bone health; improvebone health; maintain bone health; treat or manage bone disorders;support bone health; support a normal and comfortable range of motionand/or flexibility; improve range of motion and/or flexibility; reducethe action of harmful enzymes that break down bones; alter the action ofenzymes that affect bone absorption; improve movement with normal bonefunction; improve physical mobility; manage and/or maintain physicalmobility; alleviate pain and/or stiffness due to bone loss; improvephysical function; promote or enhance flexibility and comfortablemovement; promote healthy bone function and comfort; relieve bonediscomfort; relieve bone discomfort caused by exercise, work,overexertion or any combination thereof; promote healthy bones byprotecting cartilage integrity; maintain joint cartilage; support jointcartilage; treat, prevent, or manage cartilage degradation; minimizecartilage degradation; promote joint health or comfort by maintainingsynovial fluid for joint lubrication; support joint stability and jointflexibility; revitalize joints and promote mobility; promote flexiblejoints and strong cartilage; maintain steady blood flow to joints tosupport enhanced flexibility and/or strength; promote joint comfort anda wide range of motion after exercise, work, overexertion, or anycombination thereof; or any other associated indication describedherein, and generally with acceptable toxicity to a patient.

In certain other embodiments, compounds and compositions (e.g.,pharmaceutical, nutraceutical) of the present disclosure may beadministered in an amount sufficient to prevent or treat bone disorders,cartilage disorders, or both. Those osteochondrodyspiasia includesosteoporosis, osteoarthritis, osteonecrosis, osteophyte, bone fracture,metabolic bone disorders, osteochondritis diseases, osteochondroma,osteitis deformans, osteitis fibrosa cystica, ostteitis pubis,condensing osteitis, osteogenesis imperfecta, osteomalacia (rickets),osteomyelitis, osteopenia, or any other bone and cartilage associatedindication, and generally with acceptable toxicity to a patient.

Administration of the compounds of this disclosure, or theirpharmaceutically or nutraceutically acceptable salts, in pure form or inan appropriate pharmaceutical or nutraceutical composition, can becarried out via any of the accepted modes of administration of agentsfor serving similar utilities. The pharmaceutical or nutraceuticalcompositions of this disclosure can be prepared by combining a compoundof this disclosure with an appropriate pharmaceutically ornutraceutically acceptable carrier, diluent or excipient, and may beformulated into preparations in solid, semi-solid, liquid or gaseousforms, such as tablets, capsules, powders, granules, ointments,solutions, suppositories, injections, inhalants, gels, microspheres, andaerosols. Typical routes of administering such pharmaceutical ornutraceutical compositions include oral, topical, transdermal,inhalation, parenteral, sublingual, buccal, rectal, vaginal, orintranasal. The term parenteral as used herein includes subcutaneousinjections, intravenous, intramuscular, intrasternal injection orinfusion techniques. Pharmaceutical or nutraceutical compositions ofthis disclosure are formulated so as to allow the active ingredientscontained therein to be bioavailable upon administration of thecomposition to a patient. In certain embodiments, compositions of thepresent disclosure are administered to a subject or patient in the formof one or more dosage units, where, for example, a tablet may be asingle dosage unit, and a container of a compound of this disclosure inaerosol form may hold a plurality of dosage units. Actual methods ofpreparing such dosage forms are known, or will be apparent, to thoseskilled in this art; for example, see Remington: The Science andPractice of Pharmacy, 20^(th) Edition (Philadelphia College of Pharmacyand Science, 2000). The composition to be administered will, in anyevent, contain a therapeutically effective amount of a compound of thisdisclosure, or a pharmaceutically or nutraceutically acceptable saltthereof, for treatment of a disease or condition of interest inaccordance with the teachings of this disclosure.

A pharmaceutical or nutraceutical composition of this disclosure may bein the form of a solid or liquid. In one aspect, the carrier(s) areparticulate, so that the compositions are, for example, in tablet orpowder form. The carrier(s) may be liquid, with the compositions being,for example, oral syrup, injectable liquid or an aerosol, which isuseful in, for example, inhalatory administration.

When intended for oral administration, the pharmaceutical ornutraceutical composition is in either solid or liquid form, wheresemi-solid, semi-liquid, suspension and gel forms are included withinthe forms considered herein as either solid or liquid.

As a solid composition for oral administration, the pharmaceutical ornutraceutical composition may be formulated into a powder, granule,compressed tablet, pill, capsule, chewing gum, wafer, bar, or like form.Such a solid composition will typically contain one or more inertdiluents or edible carriers. In addition, one or more of the followingmay be present: binders such as carboxymethylcellulose, ethyl cellulose,cyclodextrin, microcrystalline cellulose, gum tragacanth or gelatin;excipients such as starch, lactose or dextrins, disintegrating agentssuch as alginic acid, sodium alginate, Primogel, corn starch and thelike; lubricants such as magnesium stearate or Sterotex®; glidants suchas colloidal silicon dioxide; sweetening agents such as sucrose orsaccharin; a flavoring agent such as peppermint, methyl salicylate ororange flavoring; and a coloring agent.

When the pharmaceutical or nutraceutical composition is in the form of acapsule, for example, a gelatin capsule, it may contain, in addition tomaterials of the above type, a liquid carrier such as polyethyleneglycol or oil.

The pharmaceutical or nutraceutical composition may be in the form of aliquid, for example, an elixir, syrup, gel, solution, emulsion orsuspension. The liquid may be for oral administration or for delivery byinjection, as two examples. When intended for oral administration, auseful composition contains, in addition to the present compounds, oneor more of a sweetening agent, preservatives, dye/colorant and flavorenhancer. In a composition intended to be administered by injection, oneor more of a surfactant, preservative, wetting agent, dispersing agent,suspending agent, buffer, stabilizer and isotonic agent may be included.

The liquid pharmaceutical or nutraceutical compositions of thisdisclosure, whether they be solutions, suspensions or other like form,may include one or more of the following adjuvants: sterile diluentssuch as water for injection, saline solution, such as physiologicalsaline, Ringer's solution, isotonic sodium chloride, fixed oils such assynthetic mono or diglycerides which may serve as the solvent orsuspending medium, polyethylene glycols, glycerin, propylene glycol orother solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas carbonate, citrates, acetate, lactate, gluconate, or phosphates andagents for the adjustment of tonicity such as sodium chloride ordextrose. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.Physiological saline is a generally useful adjuvant. An injectablepharmaceutical or nutraceutical composition is sterile.

A liquid pharmaceutical or nutraceutical composition of this disclosureintended for either parenteral or oral administration should contain anamount of a compound of this disclosure such that a suitable dosage willbe obtained.

The pharmaceutical or nutraceutical composition of this disclosure maybe intended for topical administration, in which case the carrier maysuitably comprise a solution, emulsion, cream, lotion, ointment, or gelbase. The base, for example, may comprise one or more of the following:petrolatum, lanolin, polyethylene glycols, bee wax, mineral oil,diluents such as water and alcohol, and emulsifiers and stabilizers.Thickening agents may be present in a pharmaceutical or nutraceuticalcomposition for topical administration. If intended for transdermaladministration, the composition may include a transdermal patch oriontophoresis device.

The pharmaceutical or nutraceutical composition of this disclosure maybe intended for rectal administration, in the form, for example, of asuppository, which will melt in the rectum and release the drug. Thecomposition for rectal administration may contain an oleaginous base asa suitable nonirritating excipient. Such bases include lanolin, cocoabutter and polyethylene glycol.

The pharmaceutical or nutraceutical composition of this disclosure mayinclude various materials, which modify the physical form of a solid orliquid dosage unit. For example, the composition may include materialsthat form a coating shell around the active ingredients. The materialsthat form the coating shell are typically inert, and may be selectedfrom, for example, sugar, shellac, and other enteric coating agents.Alternatively, the active ingredients may be encased in a gelatincapsule.

The pharmaceutical or nutraceutical composition of this disclosure insolid or liquid form may include an agent that binds to the compound ofthis disclosure and thereby assists in the delivery of the compound.Suitable agents that may act in this capacity include a monoclonal orpolyclonal antibody, a protein or a liposome.

The pharmaceutical or nutraceutical composition of this disclosure insolid or liquid form may include reducing the size of a particle to, forexample, improve bioavailability. The size of a powder, granule,particle, microsphere, or the like in a composition, with or without anexcipient, can be macro (e.g., visible to the eye or at least 100 μm insize), micro (e.g., may range from about 100 μm to about 100 nm insize), nano (e.g., may no more than 100 nm in size), and any size inbetween or any combination thereof to improve size and bulk density.

The pharmaceutical or nutraceutical composition of this disclosure mayconsist of dosage units that can be administered as an aerosol. The termaerosol is used to denote a variety of systems ranging from those ofcolloidal nature to systems consisting of pressurized packages. Deliverymay be by a liquefied or compressed gas or by a suitable pump systemthat dispenses the active ingredients. Aerosols of compounds of thisdisclosure may be delivered in single phase, bi-phasic, or tri-phasicsystems in order to deliver the active ingredient(s). Delivery of theaerosol includes the necessary container, activators, valves,subcontainers, and the like, which together may form a kit. One skilledin the art, without undue experimentation, may determine the mostappropriate aerosol(s).

The pharmaceutical or nutraceutical compositions of this disclosure maybe prepared by methodology well known in the pharmaceutical ornutraceutical art. For example, a pharmaceutical or nutraceuticalcomposition intended to be administered by injection can be prepared bycombining a compound of this disclosure with sterile, distilled water soas to form a solution. A surfactant may be added to facilitate theformation of a homogeneous solution or suspension. Surfactants arecompounds that non-covalently interact with the compound of thisdisclosure so as to facilitate dissolution or homogeneous suspension ofthe compound in the aqueous delivery system.

The compounds of this disclosure, or their pharmaceutically ornutraceutically acceptable salts, are administered in a therapeuticallyeffective amount, which will vary depending upon a variety of factorsincluding the activity of the specific compound employed; the metabolicstability and length of action of the compound; the age, body weight,general health, sex, and diet of the patient; the mode and time ofadministration; the rate of excretion; the drug combination; theseverity of the particular disorder or condition; and the subjectundergoing therapy.

Compounds of this disclosure, or pharmaceutically or nutraceuticallyacceptable derivatives thereof, may also be administered simultaneouslywith, prior to, or after administration of one or more other therapeuticagents. Such combination therapy includes administration of a singlepharmaceutical or nutraceutical dosage formulation which contains acompound of this disclosure and one or more additional active agents, aswell as administration of the compound of this disclosure and eachactive agent in its own separate pharmaceutical or nutraceutical dosageformulation. For example, a compound of this disclosure and anotheractive agent can be administered to the patient together in a singleoral dosage composition, such as a tablet or capsule, or each agent canbe administered in separate oral dosage formulations. Where separatedosage formulations are used, the compounds of this disclosure and oneor more additional active agents can be administered at essentially thesame time, i.e., concurrently, or at separate staggered times, i.e.,sequentially; combination therapy is understood to include all theseregimens.

It is understood that in the present description, combinations ofsubstituents or variables of the depicted formulae are permissible onlyif such contributions result in stable compounds.

It will also be appreciated by those skilled in the art that in theprocess described herein the functional groups of intermediate compoundsmay need to be protected by suitable protecting groups. Such functionalgroups include hydroxy, amino, mercapto and carboxylic acid. Suitableprotecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl(for example, t-butyldimethylsilyl, t-butyldiphenylsilyl ortrimethylsilyl), tetrahydropyranyl, benzyl, and the like. Suitableprotecting groups for amino, amidino and guanidino includet-butoxycarbonyl, benzyloxycarbonyl, and the like. Suitable protectinggroups for mercapto include —C(O)—R″ (where R″ is alkyl, aryl orarylalkyl), p-methoxybenzyl, trityl and the like. Suitable protectinggroups for carboxylic acid include alkyl, aryl or arylalkyl esters.Protecting groups may be added or removed in accordance with standardtechniques, which are known to one skilled in the art and as describedherein. The use of protecting groups is described in detail in Green, T.W. and P. G. M. Wutz, Protective Groups in Organic Synthesis (1999),3^(rd) Ed., Wiley. As one of skill in the art would appreciate, aprotecting group may also be a polymer resin such as a Wang resin, Rinkresin or a 2-chlorotrityl-chloride resin.

It will also be appreciated by those skilled in the art, although suchprotected derivatives of compounds of this disclosure may not possesspharmacological activity as such, they may be administered to a mammaland thereafter metabolized in the body to form compounds of thisdisclosure which are pharmacologically active. Such derivatives maytherefore be described as “prodrugs”. All prodrugs of compounds of thisdisclosure are included within the scope of this disclosure.

Furthermore, all compounds of this disclosure which exist in free baseor acid form can be converted to their pharmaceutically ornutraceutically acceptable salts by treatment with the appropriateinorganic or organic base or acid by methods known to one skilled in theart. Salts of the compounds of this disclosure can be converted to theirfree base or acid form by standard techniques.

In some embodiments, compounds of the present disclosure can be isolatedfrom plant sources, for example, from those plants included in theExamples and elsewhere throughout the present application. Suitableplant parts for isolation of the compounds include leaves, bark, trunk,trunk bark, stems, stem bark, twigs, tubers, root, root bark, barksurface (such as periderm or polyderm, which may include phellem,phellogen, phelloderm, or any combination thereof), young shoots,rhizomes, seed, fruit, androecium, gynoecium, calyx, stamen, petal,sepal, carpel (pistil), flower, or any combination thereof. In somerelated embodiments, the compounds are isolated from plant sources andsynthetically modified to contain any of the recited substituents. Inthis regard, synthetic modification of the compound isolated from plantscan be accomplished using any number of techniques that are known in theart and are well within the knowledge of one of ordinary skill in theart.

Morus alba L (Moraceae), the mulberry or white berry plant, is native tonorthern China, and has been cultivated and naturalized elsewhere, fromIndia to the Middle East to Southern Europe, and recently to the NorthAmerican area. Morus root-bark is used in traditional medicine known asSang bai pi or Cortex Mori (Pharmacopoeia of the People's Republic ofChina, 2005). Morus herb is also known as Pong-na-moo in Korean andSohakuhi in Japan. In contemporary pharmacological research, Morus albaroot-bark has been reported to have antibacterial, anti-viral,antioxidant, hypoglycemic, hypolipidemic, neuroprotective, antiulcer,analgesic and anti-inflammatory activities. A variety of bioactivecompounds from Morus alba root-bark have in vivo and in vitroanti-inflammatory activity.

As noted herein, compounds of a Diels-Alder adduct of a chalcone and aprenylphenyl moiety, prenylated flavonoids, stilbenes, or anycombination thereof may be obtained by chemical synthesis or from aplant extract, such as a Morus or Milicia extract. For example, Morus isa genus of flowering trees in the family Moraceae, which comprises morethan 30 species (known as mulberries) that grow wild or undercultivation in many countries. Exemplary Morus species include Morusalba L., Morus australis Poir, Morus celtidifolia Kunth, Morus insignis,Morus mesozygia Stapf, Morus microphylla, Morus nigra L., Morus rubraL., Morus atropurpurea, Morus bombycis, Morus cathayana, Morus indica,Morus thou, Morus japonica, Morus kagayamae, Morus laevigata, Moruslatifolia, Morus liboensis, Morus macroura, Morus mongolica, Morusmulticaulis, Morus notabilis, Morus rotundiloba, Morus serrate, Morusheterophyllus, Morus tillaefolia, Morus trilobata, Morus yunnanensis,and Morus wittiorum.

In certain embodiments, a Morus extract is from Morus alba, or a Morusextract is a mixture of extracts from one, two, three, four, or fivedifferent Morus species. A mixture of extracts may include extracts fromtwo or more Morus species or other sources listed in Table A. Forexample, a composition comprising a Diels-Alder adduct of a chalcone anda prenylphenyl moiety, a prenylated flavonoid, a stilbene, or anycombination thereof may be made up of a Morus extract (e.g., Morus alba)and a Milicia extract (e.g., Milicia excelsa). In certain embodiments, aMorus extract enriched for prenylated flavonoids and stilbenes is fromMorus alba (a) root bark, (b) root bark and leaves, (c) rootbark andtwigs, (d) root bark, leaves and twigs, or (e) root bark, root wood,fine roots, stem bark, branch, branch bark, branch wood, and twigs.

In some specific embodiments, compounds of a Diels-Alder adduct of achalcone and a prenylphenyl moiety may be any one or more of thecompounds provided in Table A.

TABLE A List of Exemplary Diels-Alder Adduct Compounds MolecularStructure Name Species Formula M.W.

Albafuran C Morus alba C₃₄H₂₈O₉ 580.590

Albafuran C; 2- Epimer Morus australis C₃₄H₂₈O₉ 580.590

Albanin F Morus alba, also from Morus australis, Morus bombycis, andMorus lhou C₄₀H₃₆O₁₁ 692.718

Albanin F (Moracenin D); 12,13-Dihydro, 13- hydroxy Morus sp. C₄₀H₃₈O₁₂710.733

Albanin G (Kuwanon H. Moracenin A.) Morus alba; also isol. from Morusaustralis, Morus bombycis, and Morus lhou C₄₅H₄₄O₁₁ 760.836

Albanin G; 2′′′- Deoxy (Mongolicin D) Morus mongolica C₄₅H₄₄O₁₀ 744.837

Albanol A (Mulberrofuran G.) Morus lhou C₃₄H₂₆O₈ 562.575

Albanol A; 3″-(3- Methyl-2-butenyl), Mulberrofuran F Morus lhou C₃₉H₃₄O₈630.693

Albanol B Morus alba C₃₄H₂₂O₈ 558.543

Artonin C Artocarpus heterophyllus (jackfruit) C₄₀H₃₈O₁₀ 678.734

Artonin D Artocarpus heterophyllus (jackfruit) C₄₀H₃₆O₁₀ 676.718

Artonin I Morus heterophyllus C₄₀H₃₆O₁₁ 692.718

Australisin B Morus australis C₃₉H₃₄O₉ 646.692

Australisin C.; 2- Epimer Morus australis C₃₄H₂₈O₉

Brosimone B Brosimopsis oblongifolia (preferred genus name Brosimum)C₄₀H₃₈O₁₀ 678.734

Brosimone D Brosimopsis oblongifolia (preferred genus name Brosimum)C₄₅H₄₄O₁₁ 760.836

Cathayanon A Morus cathayana C₄₀H₃₆O₁₂ 708.717

Cathayanon A; 14- Epimer Morus cathayana C₄₀H₃₆O₁₂ 708.717

Cathayanon E Morus cathayana C₄₀H₃₆O₁₂ 708.717

Chalcomoracin Morus alba and Morus mongolica C₃₉H₃₆O₉ 648.708

Chalcomoracin; 3″,5″-Diepimer Sorocea muriculata C₃₉H₃₆O₉ 648.708

Chalcomoracin; 3″-Epimer Morus mongolica C₃₉H₃₆O₉ 648.708

Dorstenone Dorstenia barteri C₄₀H₃₈O₈ 646.735

Guangsangon C Morus macroura C₃₅H₃₀O₁₀ 610.616

Guangsangon D Morus macroura C₃₅H₃₀O₁₀ 610.616

Guangsangon D; 2′-Deoxy, 4′,6′- dihydroxy Morus macroura C₃₅H₃₀O₁₁626.615

Guangsangon D; 3- Deoxy, 4′-hydroxy Morus macroura and Morus wittiorumC₃₅H₃₀O₁₀ 610.616

Guangsangon D; 2- Epimer, 3-deoxy, 4′-hydroxy Morus macroura C₃₅H₃₀O₁₀610.616

Guangsangon E Morus macroura C₃₉H₃₆O₉ 648.708

Guangsangon E; 3″-Epimer, 2′′′′,3′′′′-dihydro, 3′′′′-hydroxy Morusmacroura C₃₉H₃₈O₁₀ 666.723

Guangsangon F Morus macroura C₄₀H₃₆O₁₀ 676.718

Guangsangon G Morus macroura C₃₅H₂₈O₁₀ 608.600

Guangsangon G; 1″-Epimer, 2′- hydroxy Morus macroura C₃₅H₂₈O₁₁ 624.600

Guangsangon G; 2′-Hydroxy Morus macroura C₃₅H₂₈O₁₁ 624.600

Guangsangon G; 5- Hydroxy Morus wittiorum C₃₅H₂₈O₁₁ 625.600

Guangsangon H Morus macroura C₄₀H₃₈O₁₀ 678.734

Guangsangon J Morus macroura C₃₉H₃₆O₉ 648.708

Guangsangon L Morus alba C₂₇H₂₄O₈ 476.482

Isobavachromene dimer Dorstenia zenkeri C₄₀H₃₈O₈ 646.735

Kuwanol A Morus bombycis C₃₄H₂₈O₈ 564.590

Kuwanol B Morus bombycis C₃₄H₂₆O₈ 562.575

Kuwanol E Morus alba (white mulberry) C₃₉H₃₈O₉ 650.724

Kuwanol E; 2′′′,3′′′-Dihydro, 3′′′-hydroxy Sorocea ilicifolia C₃₉H₄₀O₁₀668.739

Kuwanon J Morus alba and from Morus bombycus and Morus nigra C₄₀H₃₈O₁₀678.734

Kuwanon J; 16″- Deoxy Morus alba (white mulberry) C₄₀H₃₈O₉ 662.735

Kuwanon J; 2- Deoxy Morus alba (white mulberry) C₄₀H₃₈O₉ 662.735

Kuwanon J, Δ21″,22″-Isomer, 2-deoxy Morus alba (white mulberry) C₄₀H₃₈O₉662.735

Kuwanon J; 2,16″- Dideoxy Morus alba (white mulberry) C₄₀H₃₈O₈ 646.735

Kuwanon J; 2′,3′- Dihydro Morus mongolica C₄₀H₄₀O₁₀ 680.750

Kuwanon J; 1″- Epimer Morus alba and Morus bombycus C₄₀H₃₈O₁₀ 678.734

Kuwanon J; Δ21″,22″-Isomer, 2-deoxy (Artonin X.) Artocarpusheterophyllus (jackfruit) C₄₀H₃₈O₉ 662.735

Kuwanon L Morus alba (white mulberry) C₃₅H₃₀O₁₁ 626.615

Kuwanon L; 2,3- Didehydro, 3-(3- methyl-2-butenyl) Morus alba (whitemulberry) C₄₀H₃₆O₁₁ 692.718

Kuwanon N Morus lhou C₄₅H₄₄O₁₁ 760.836

Kuwanon O Morus lhou C₄₀H₃₈O₁₁ 694.734

Kuwanon P Morus lhou C₃₄H₃₀O₉ 582.606

Kuwanon P; 2- Deoxy Morus macroura C₃₄H₃₀O₈

Kuwanon W Morus lhou C₄₅H₄₂O₁₁ 758.820

Kuwanon X Morus lhou C₃₄H₃₀O₉ 582.606

Kuwanon X; 3″- Epimer Morus alba (white mulberry) C₃₄H₃₀O₉ 582.606

Kuwanon Z Morus alba (white mulberry) C₃₄H₂₆O₁₀ 594.573

Mongolicin C Morus mongolica C₃₄H₂₆O₉ 578.574

Moracenin C Morus sp. C₄₅H₄₄O₁₁ 760.836

Mulberrofuran C Morus bombycis (Moraceae)

Mulberrofuran E Morus alba (white mulberry) (Moraceae) C₃₉H₃₆O₈ 632.709

Mulberrofuran I Morus bombycis C₃₄H₂₄O₈ 560.559

Mulberrofuran J Morus lhou C₃₄H₂₈O₉ 580.590

Mulberrofuran J, 2-Epimer Morus bombycis

Mulberrofuran O Morus alba 646.692

Mulberrofuran P Morus alba (white mulberry) C₃₄H₂₂O₉ 574.542

Mulberrofuran Q Morus alba (white mulberry) C₃₄H₂₄O₁₀ 592.558

Mulberrofuran S Morus alba (white mulberry) C₃₄H₂₄O₉ 576.558

Mulberrofuran T Morus alba (white mulberry) C₄₄H₄₄O₉ 716.826

Mulberrofuran U Morus insignis C₃₉H₃₆O₉ 648.708

Multicaulisin Morus multicaulis C₄₀H₃₆O₁₁ 692.718

Sanggenol G Morus cathayana C₃₀H₃₄O₇ 694.734

Sanggenol J Morus cathayana C₄₅H₄₄O₁₂ 776.835

Sanggenol M Morus mongolica C₄₄H₄₄O₁₁ 748.825

Sanggenon B Morus C₃₃H₃₀O₉ 570.595

Sanggenon B; 7-O- (2,4- Dihydroxybenzoyl) (Sanggenon S) Morus spC₄₀H₃₄O₁₂ 706.701

Sanggenon D Morus cathayana C₄₀H₃₆O₁₂ 708.717

Sanggenon E Morus Spp. C₄₅H₄₄O₁₂ 776.835

Sanggenon G Morus alba C₄₀H₃₈O₁₁ 694.734

Sanggenon G; 14,15-Dihydro, 15- hydroxy Morus sp. C₄₀H₄₀O₁₂ 712.749

Sanggenon Q Morus mongolica C₄₀H₃₆O₁₂ 708.717

Sanggenon D; 3′- Epimer Morus cathayana C₄₀H₃₆O₁₂ 708.717

Sanggenon D; 2,3,3′-Triepimer Morus cathayana C₄₀H₃₆O₁₂ 708.717

Sorocein B Sorocea bonplandii C₄₀H₃₄O₉ 658.703

Sorocein H Sorocea bonplandii (Moraceae) and Morus spp. C₄₅H₄₄O₁₂776.835

Wittiorumin B Morus wittiorum C₄₀H₃₆O₁₂ 708.717

Wittiorumin B; 1″- Epimer, 2′-deoxy Morus wittiorum C₄₀H₃₆O₁₁ 692.718

Wittiorumin E Morus wittiorum C₄₀H₃₈O₁₀ 678.734

Wittiorumin F Morus wittiorum C₃₉H₃₆O₉ 648.708

Wittiorumin G Morus wittiorum C₄₀H₃₈O₁₀ 678.734

Yunanensin A Morus yannanensis C₃₉H₂₈O₈ 624.645

Compounds in Table A and Examples 3, 5, and 6 can be extracted, isolatedor purified from the indicated plant species or certain plant parts(e.g., from the bark, trunk, trunk bark, stem bark, root, root bark,bark surface (such as periderm or polyderm, which may include phellem,phellogen, phelloderm, or any combination thereof), leaves, fruits,flowers, other plant parts, or any combination thereof) or can beprepared synthetically or semi-synthetically as described in more detailin PCT Application No. PCT/US2013/43188, which methods of synthesis areincorporated herein by reference. In certain embodiments, one or morecompounds of Table A and Examples 3, 5, and 6 are enriched for or arethe major active ingredients in an extract of the indicated plantspecies, wherein the enriched extract is obtained from a whole plant orcertain plant parts, such as leaves, bark, trunk, trunk bark, stems,stem bark, twigs, tubers, root, root bark, bark surface (such asperiderm or polyderm, which may include phellem, phellogen, phelloderm,or any combination thereof), young shoots, rhizomes, seed, fruit,androecium, gynoecium, calyx, stamen, petal, sepal, carpel (pistil),flower, or any combination thereof.

In further embodiments, major active ingredients in an extract of Moruscomprise prenylated flavonoids and stilbenes (such as those provided inTable A and Examples 3, 5, and 6), wherein the extract is enriched forthese active ingredients from root bark, leaves, twigs, or a combinationthereof. In certain embodiments, a Morus extract is enriched forprenylated flavonoids and stilbenes, wherein the extract comprises fromabout 1% to about 25% prenylated flavonoids and from about 1% to about25% stilbenes, or wherein the extract comprises from about 2% to about6% prenylated flavonoids and from about 2% to about 6% stilbenes, orwherein the extract comprises at least 3% prenylated flavonoids and atleast 3% stilbenes (weight to weight).

In certain embodiments, provided herein are Morus extracts enriched forone or more prenylated flavonoids or chalconoids and one or morestilbenes, wherein the one or more prenylated flavonoids are compoundshaving a structure of Formula (III) or (IV):

or a pharmaceutically or nutraceutically acceptable salt, tautomer,glycoside, prodrug or stereoisomer thereof, wherein R₁-R₁₂ are eachindependently H, hydroxyl, a prenyl group, flavonoid, chalcone,glycoside, halogen, sulfhydryl, amino, aldehyde, C₁₋₁₂ alkyl, C₁₋₁₂alkoxy, C₁₋₁₂ alkthio, C₁₋₁₂ alkyamino, cycloalkyl, heterocyclyl, aryl,heteroaryl, aralkyl, alkyl carbonyl, aralkylcarbonyl or a bond to acompound of structure (III) or (IV); or one of R₁-R₁₂ joins with anotherone of R₁-R₁₂ to form a ring, and the remaining R₁-R₁₂ are eachindependently H, hydroxyl, a prenyl group, flavonoid, chalcone,glycoside, halogen, sulfhydryl, amino, aldehyde, C₁₋₁₂ alkyl, C₁₋₁₂alkoxy, C₁₋₁₂ alkthio, C₁₋₁₂ alkyamino, cycloalkyl, heterocyclyl, aryl,heteroaryl, aralkyl, alkylcarbonyl, aralkylcarbonyl or a bond to acompound of structure (III) or (IV), provided that all valencies aresatisfied; the chalcanoid is a compound of structure (V):

or a pharmaceutically or nutraceutically acceptable salt, tautomer,glycoside, prodrug or stereoisomer thereof, wherein R₁-R₁₀ are eachindependently H, hydroxyl, a prenyl group, flavonoid, chalcone,glycoside, halogen, sulfhydryl, amino, aldehyde, C₁₋₁₂ alkyl, C₁₋₁₂alkoxy, C₁₋₁₂ alkthio, C₁₋₁₂ alkyamino, cycloalkyl, heterocyclyl, aryl,heteroaryl, aralkyl, alkyl carbonyl, or aralkylcarbonyl, provided thatall valencies are satisfied; and the one or more stilbenes are compoundshaving a structure of Formula (I) or (II):

wherein R₁-R₁₀ are each independently a H, hydroxyl, glycoside, prenyl,flavonoid, chalcone, halogen, sulfhydryl, amino, aldehyde, C₁₋₁₂ alkyl,C₁₋₁₂ alkenyl, C₁₋₁₂ alkoxy, C₁₋₁₂ alkthio, C₁₋₁₂ alkyamino, aryl,heteroaryl, aralkyl, alkylcarbonyl, or aralkylcarbonyl.

In further embodiments, the one or more prenylayted flavonoids arecompounds having a structure of Formula (III), (IV) or (V), wherein theoptional double bond is present in ring C, R₁₁ and R₁₂ are absent, andR₁₀ is a prenyl group. In still further embodiments, the one or moreprenylayted flavonoids are compounds having a structure of Formula(III), (IV) or (V), wherein the at least one of R₁-R₉ is a prenyl groupand R₁₀-R₁₂ are independently H or hydroxyl. In certain specificembodiments, the prenylated flavonoids include Albanin G, Kuwanon G,Morusin, morusinol, Sanggenon, isoxanthoumol, glabridin, cathayanon A,or any combination thereof. In certain embodiments, the one or morestilbenes are compounds having a structure of Formula (I) or (II),wherein R₁-R₁₀ are each independently a H, hydroxyl, glycoside, or C₁₋₄alkoxy. In further embodiments, the one or more stilbenes are compoundshaving a structure of Formula (I) or (II), wherein R₁, R₅, R₆ and R₁₀are H. In still further embodiments, the one or more stilbenes arecompounds having a structure of Formula (I) or (II), wherein R₂ is aglucoside, or R₂ and R₈ are glycosides, and one or more of R₄, R₉, andR₁₀ are hydroxyl. In yet further embodiments, the one or more stilbenesare compounds having a structure of Formula (I) or (II), wherein R₁, R₅,and R₆ are H, and one or more of R₂-R₄ and R₇-R₁₀ are independentlyhydroxyl, C₁₋₃ alkoxy, or any combination thereof. In certain specificembodiments, a stilbene compound includes oxyresveratrol, resveratrol,piceatannol, pinosylvin, 3,4′-dihydroxystilbene, combretastatin A-1,pterostilbene, rhapontigenin, and a stilbene glycoside includesmulberroside A, rhaponticin, piceid, astringin, or any combination ofthese stilbenes or stilbene glycosides.

In some embodiments, the flavonoid is a compound of structure (III) andin other embodiments the flavonoid is a compound of structure (IV). Insome of the embodiments, at least one of R₁-R₁₂, such as R₁₀ is prenyl.In other embodiments, polyflavonoids are provided and at least one ofR₁-R₁₂ in the compounds of structure (III) or (IV) is a bond to acompounds of structure of (III) or (IV) (i.e., the compound comprisesmore than one flavonoid of structure (III) and/or (IV)).

In some other embodiments of the compounds of structure (III) or (IV),R₁-R₁₂ is H, hydroxyl, a prenyl group or cycloalkyl. For example, insome embodiments the cycloalkyl is substituted and/or comprises one ormore carbon-carbon double bonds (i.e., is unsaturated). The optionalsubstitutents are typically selected from aryl, such as phenyl, and arylcarbonyl. Accordingly, in some further embodiments, the flavonoid hasone of the following structures (IIIa) or (IVa):

wherein R^(4a) is, at each occurrence, independently H, hydroxyl or aprenyl group.

In certain embodiments of the compounds of structure (IIIa) or (IVa),R₁-R₃ and R₅-R₁₂ are each independently selected from H, hydroxyl and aprenyl group. In certain embodiments, at least one one of R₁-R₃, R_(4a)or R₅-R₁₂ is prenyl, for example in some embodiments, R₁₀ is prenyl. Inother embodiments of the compounds of structure (IIIa) or (IVa), atleast two of R₁-R₃, R_(4a) or R₅-R₁₂ is hydroxyl.

In some more specific embodiments, the flavonoid has one of thefollowing structures:

In other embodiments, one of R₁-R₁₂ joins with another one of R₁-R₁₂ toform a ring and the remaining R₁-R₁₂ are H, hydroxyl or a prenyl group.In certain of these embodiments, the ring is a heterocyclic ring, forexample a cyclic ether ring. Accordingly, in certain embodiments theflavonoid has one of the following structures (IIIb) or (IVb):

In certain embodiments of the compounds of structure (IIIb) or (IVb),R₁, R₂ and R₅-R₁₂ are each independently selected from H, hydroxyl and aprenyl group. In certain embodiments, at least one one of R₁, R₂ orR₅-R₁₂ is prenyl, for example in some embodiments, R₁₀ is prenyl. Inother embodiments of the compounds of structure (IIIb) or (IVb), atleast two of R₁, R₂ or R₅-R₁₂ is hydroxyl. In certain embodiments, theflavonoid has the following structure:

In various other embodiments, R₁-R₁₀ of the chalcanoid of structure (V)are each independently selected from H, hydroxyl, a prenyl group, andC₁₋₁₂ alkoxy.

Acacia is a genus of leguminous trees and shrubs. The genus Acaciaincludes more than 1000 species belonging to the family of Leguminosaeand the subfamily of Mimosoideae. Acacias are distributed worldwide intropical and subtropical areas of Central and South America, Africa,parts of Asia, as well as Australia, which has the largest number ofendemic species. Acacias occur primarily in dry and arid regions, wherethe forests are often in the nature of open thorny shrubs. Acacias arevery important economically, providing a source of tannins, gums,timber, fuel and fodder. Tannins, which are isolated primarily frombark, are used extensively for tanning hides and skins Some Acacia barksare also used for flavoring local spirits. Some indigenous species likeA. sinuata also yield saponins, which are used in detergents, foamingagents and emulsifiers. The flowers of some Acacia species are fragrantand used to make perfume. The heartwood of many Acacias is used formaking agricultural implements and also provides a source of firewood.Acacia gums find extensive use in medicine and confectionary and assizing and finishing materials in the textile industry.

Uncaria gambir (Rubiaceae) is a climbing shrub with round branches,which is believed to strengthen teeth when chewed with piper bettleleaves. All parts of the plant have astringent properties. Leaves of theU. gambir plant contain free catechins as well as polymerizedcatechins—tannins—which are more abundant in younger leaves as comparedto older leaves. U. gambir is listed in the Food Additive Database inEAFUS (Everything Added to Food in the United States), in the Korea FoodAdditives Code by KFDA, and in the Japan Food Additives Code by MHLW asa natural flavoring agent. U. gambir is also listed in the KoreaPharmaceutical Codex (KP), Japan Pharmaceutical Codex (JP) and ChinaPharmaceutical Codex (CP). In South Korea, there are manyover-the-counter (OTC) drugs that contain U. gambir extract, especiallyfor dyspepsia, halitosis, vomiting and anorexia. In Japan, U. gambir isused for diarrhea, vomiting and gastritis. In the United States, U.gambir is used as a dietary supplement to support liver function and fatmetabolism.

The biologically active flavans of this disclosure may be obtained bysynthetic methods or extracted from one or more plants, such as Acacia,Uncaria, or both. In certain embodiments, an Acacia plant species isselected from A. angustifolia, A. ataxacantha, A. berlandieri, A.bonariensis, A. brevispica, A. catechu, A. chundra, A. concinna, A.floribunda, A. greggii, A. interior, A. macilenta, A. mellifera, A.merrallii, A. occidentalis, A. peninsularis, A. pennata, A. pennatula,A. polyacantha, A. polyphylla, A. riparia, A. roemeriana, A. senegal, A.sinuata, A. tamarindifolia, A. tenuifolia, A. victoriae, A. visco, orany any combination thereof (for exemplary Acacia extracts and flavans,see U.S. Pat. No. 8,124,134). In certain embodiments, an Uncaria plantspecies is selected from U. acida, U. africana, U. attenuate, U.bernaysii, U. borneensis, U. callophylla, U. cordata, U. elliptica,Uncaria gambir, U. guianensis, U. hirsute, U. homomalla, U. lanosa, U.longiflora, U. macrophylla, U. orientalis, U. rhynchophylla, U.scandens, U. sessilifructus, U. setiloba, U. sinensis, U. sterrophylla,U. tomentosa, U. wangii, or any any combination thereof (for exemplaryUncaria extracts and flavans, see U.S. Patent Publication No.2007/0264361).

In further embodiments, a composition of this disclosure comprises anAcacia catechu extract enriched for flavans containing catechin,epicatechin, or a combination thereof. In still further embodiments, acomposition of this disclosure comprises an Uncaria gambir extractenriched for flavans containing catechin, epicatechin, or a combinationthereof. In yet further embodiments, an Acacia extract enriched forflavans is from Acacia catechu, or an Acacia extract enriched forflavans is a mixture of extracts from one, two, three, four, five ormore different Acacia species, Uncaria species, or from other sources.In other embodiments, an Uncaria extract enriched for flavans is fromUncaria gambir, or an Uncaria extract enriched for flavans is a mixtureof extracts from one, two, three, four, five or more different Uncariaspecies, Acacia species, other sources (e.g., different plant such asgreen tea, synthetic), or any combination thereof. For example, acomposition of this disclosure comprises a mixture of an Acacia catechuextract enriched for flavans containing catechin, epicatechin, or bothand an Uncaria gambir extract enriched for flavans containing catechin,epicatechin, or both.

In certain embodiments, major active ingredients in an extract of Acaciacomprise flavans containing catechin, epicatechin, or both, wherein theextract is enriched for these active ingredients from roots, bark, or acombination thereof. In certain embodiments, major active ingredients inan extract of Uncaria comprise flavans containing catechin, epicatechin,or both, wherein the extract is enriched for these active ingredientsfrom leaves.

In certain embodiments, provided herein are Acacia or Uncaria extractsenriched for one or more flavans containing catechin, epicatechin, orboth, wherein the flavans are compounds having a structure of Formula(VI):

wherein R₂₁, R₂₂, R₂₃, R₂₄ and R₂₅ are independently selected from a H,—OH, —SH, —OCH₃, —SCH₃, —OR, —SR, —NH₂, —NRH, —NR₂, —NR₃ ⁺X⁻, esters ofsubstitution groups, independently selected from the group consisting ofgallate, acetate, cinnamoyl and hydroxyl-cinnamoyl esters,trihydroxybenzoyl esters and caffeoyl esters; a carbon, oxygen, nitrogenor sulfur glycoside of a single or a combination of multiple sugarsincluding aldopentose, methyl aldopentose, aldohexose, ketohexose;dimer, trimer or other polymerized flavans;

wherein R is a C₁₋₁₀ alkyl group; and

X is a pharmaceutically acceptable counter anion of hydroxyl, chloride,iodide, sulfate, phosphate, acetate, fluoride, or carbonate.

Curcuma longa L, with common name as turmeric, is a perennial plant ofthe ginger family, Zingiberaceae. The name of turmeric might come fromLatin, terra merita (merited earth) or turmeryte, which is related tosaffron. It is originally from tropical south Asia and cultivatedextensively in India and Southeast Asia. Turmeric is prepared from theground rhizome and has been used in India for thousands of years.Besides its culinary usage, modern research has revealed that turmerichas antibacterial, antioxidant, chemopreventive, chemotherapeutic,antiproliferative, antiparasitic, anti-antimalarial, antinociceptive,and anti-inflammatory properties.

In certain embodiments, there are provided herein Curcuma extractscomprising curcuminoids. In further embodiments, a Curcuma longa extractis enriched for curcuminoids, such as curcumin (diferuloylmethane),demethoxy-curcumin, bisdemethoxy-curcumin, casumunin A, cassumunin B, orany combination thereof. The biologically active curcuminoids andanalogues therof of this disclosure may be obtained by synthetic methods(see Anand et al., Biochem. Pharmacol. 76:1590, 2008) or extracted fromone or more plants, such as Curcuma plants, Zingiber plants, or both.

Exemplary species of the Curcuma genus of the instant disclosure includeC. aeruginosa, C. albicoma, C. albiflora, C. alismatifolia, C. amada, C.amarissima, C. americana, C. angustifolia, C. aromatics, C. attenuata,C. aurantiaca, C. australasica, C. bakeriana, C. bicolor, C. bhatii, C.brog, C. burttii, C. caesia, C. candida, C. cannanorensis, C. caulina,C. careyana, C. ceratotheca, C. chuanezhu, C. chuanhuangjiang, C.chuanyujin, C. coccinea, C. cochinchinensis, C. codonantha, C. coerulea,C. colorata, C. comosa, C. cordata, C. cordifolia, C. coriacea, C.decipiens, C. domestica, C. ecalcarata, C. ecomata, C. elata, C.erubescens, C. euchroma, C. exigua, C. ferruginea, C. flaviflora, C.glans, C. glaucophylla, C. gracillima, C. grahamiana, C. grandiflora, C.haritha, C. harmandii, C. heyneana, C. inodora, C. karnatakensis, C.kuchoor, C. kudagensis, C. kiinstleri, C. kurzii, C. kwangsiensis, C.lanceolata, C. larsenii, C. latiflora, C. latifolia, C. leucorhiza, C.leucorrhiza, C. loerzingii, C. longa, C. longiflora, C. longispica, C.lutea, C. malabarica, C. mangga, C. meraukensis, C. montana, C. musacea,C. mutabilis, C. neilgherrensis, C. nilamburensis, C. ochrorhiza, C.officinalis, C. oligantha, C. ornata, C. pallida, C. parviflora, C.parvula, C. peethapushpa, C. petiolata, C. phaeocaulis, C. picta—C.pierreana, C. plicata, C. porphyrotaenia, C. prakasha, C. pseudomontana,C. purpurascens, C. purpurea, C. raktakanta, C. ranadei, C. reclinata,C. rhabdota, C. rhomba, C. roscoeana, C. rotunda, C. rubescens, C.rubricaulis, C. rubrobracteata, C. sattayasaii, C. sessilis, C.sichuanensis, C. singularis, C. soloensis, C. sparganiifolia, C.speciosa, C. spicata, C. stenochila, C. strobilifera, C. sulcata, C.sumatrana, C. sylvatica, C. sylvestris, C. thalakaveriensis, C.thorelii, C. trichosantha, C. vamana, C. vellanikkarensis, C.viridiflora, C. vitellina C. wenchowensis, C. wenyujin, C. xanthorrhiza,C. yunnanensis, C. zedoaria, C. zedoaroides, C. zerumbet.

In certain embodiments, a Curcuma extract enriched for curcuminoids isfrom Curcuma longa, or a Curcuma extract enriched for curcuminoids is amixture of extracts from one, two, three, four, five or more differentCurcuma species or from other sources. For example, a compositioncomprising curcuminoids may be a a Curcuma extract (e.g., Curcuma longa)mixed with synthetic curcuminoids, or a mixture of a Curcuma extract(e.g., Curcuma longa) enriched for curcuminoids with a Zingibercassumunar extract enriched for curcuminoids, Curcuma phaeocaulisextract enriched for curcuminoids, Curcuma. xanthorrhiza extractenriched for curcuminoids, or any combination thereof. In otherembodiments, a Curcuma extract enriched for one or more curcuminoids(e.g., curcumin, demethoxy-curcumin, bisdemethoxy-curcumin, casumunin A,cassumunin B, or any combination thereof) may be from root, rhizome, ora combination thereof.

A Morus extract enriched for prenylated flavonoids and stilbenes may beused as an anti-inflammatory by inhibiting, for example, both COX andLOX pathways, which can be utilized as is or in combination with atleast one bioactive plant extract, such as an extract from Acacia,Uncaria, Curcuma or a combination thereof, and optionally contain apharmaceutically or nutraceutically acceptable active, adjuvant,carrier, diluent, or excipient. In certain embodiments, any of theaforementioned compositions can be used to prevent bone loss, increasebone density, prevent, manage or treat osteoporosis in a mammal, such asa human, especially pre-menopausal, menopausal and post-menopausalwomen.

In certain embodiments, a composition of this disclosure comprises anAcacia extract containing or enriched for one or more flavans asdescribed herein (or described in U.S. Pat. No. 8,124,134), and a Morusextract containing or enriched for at least one Diels-Alder adduct of achalcone and a prenylphenyl moiety, prenylated flavonoid, stilbene, orany combination thereof. In certain embodiments, a composition comprisesan Acacia extract containing or enriched for one or more flavans asdescribed herein or in U.S. Pat. No. 8,124,134 and a Morus extractcontaining or enriched for one or more compounds listed in Table A andExamples 3, 5, 6 and 68. In still further embodiments, a compositioncomprises an Acacia extract containing or enriched for catechin,epicatichin, or both, and a Morus extract containing or enriched for oneor more prenylated flavonoids, one or more stilbenes, or any combinationthereof. In other embodiments, a composition comprises a mixture of aMorus extract enriched for one or more prenylated flavonoids and one ormore stilbenes, and an Acacia extract enriched for flavans.

In further embodiments, a composition of this disclosure comprises amixture of a Morus extract enriched for one or more prenylatedflavonoids and one or more stilbenes, and an Acacia extract enriched forone or more flavans,

wherein the one or more prenylated flavonoids are compounds having astructure of Formula (III) or (IV):

or a pharmaceutically or nutraceutically acceptable salt, tautomer,glycoside, prodrug or stereoisomer thereof, wherein R₁-R₁₂ are eachindependently H, hydroxyl, a prenyl group, flavonoid, chalcone,glycoside, halogen, sulfhydryl, amino, aldehyde, C₁₋₁₂ alkyl, C₁₋₁₂alkoxy, C₁₋₁₂ alkthio, C₁₋₁₂ alkyamino, cycloalkyl, heterocyclyl, aryl,heteroaryl, aralkyl, alkyl carbonyl, aralkylcarbonyl or a bond to acompound of structure (III) or (IV); or one of R₁-R₁₂ joins with anotherone of R₁-R₁₂ to form a ring, and the remaining R₁-R₁₂ are eachindependently H, hydroxyl, a prenyl group, flavonoid, chalcone,glycoside, halogen, sulfhydryl, amino, aldehyde, C₁₋₁₂ alkyl, C₁₋₁₂alkoxy, C₁₋₁₂ alkthio, C₁₋₁₂ alkyamino, cycloalkyl, heterocyclyl, aryl,heteroaryl, aralkyl, alkylcarbonyl, aralkylcarbonyl or a bond to acompound of structure (III) or (IV), provided that all valencies aresatisfied;

the chalcanoid is a compound of structure (V):

or a pharmaceutically or nutraceutically acceptable salt, tautomer,glycoside, prodrug or stereoisomer thereof, wherein R₁-R₁₀ are eachindependently H, hydroxyl, a prenyl group, flavonoid, chalcone,glycoside, halogen, sulfhydryl, amino, aldehyde, C₁₋₁₂ alkyl, C₁₋₁₂alkoxy, C₁₋₁₂ alkthio, C₁₋₁₂ alkyamino, cycloalkyl, heterocyclyl, aryl,heteroaryl, aralkyl, alkylcarbonyl, or aralkylcarbonyl, provided thatall valencies are satisfied; and

the one or more stilbenes are compounds having a structure of Formula(I) or (II):

wherein R₁-R₁₀ are each independently a H, hydroxyl, glycoside, prenyl,flavonoid, chalcone, halogen, sulfhydryl, amino, aldehyde, C₁₋₁₂ alkyl,C₁₋₁₂ alkenyl, C₁₋₁₂ alkoxy, C₁₋₁₂alkthio, C₁₋₁₂ alkyamino, aryl,heteroaryl, aralkyl, alkylcarbonyl, or aralkylcarbonyl; and wherein theflavans are compounds having a structure of Formula (VI):

wherein R₂₁, R₂₂, R₂₃, R₂₄ and R₂₅ are independently selected from a H,—OH, —SH, —OCH₃, —SCH₃, —OR, —SR, —NH₂, —NRH, —NR₂, —NR₃ ⁺X⁻, esters ofsubstitution groups, independently selected from the group consisting ofgallate, acetate, cinnamoyl and hydroxyl-cinnamoyl esters,trihydroxybenzoyl esters and caffeoyl esters; a carbon, oxygen, nitrogenor sulfur glycoside of a single or a combination of multiple sugarsincluding aldopentose, methyl aldopentose, aldohexose, ketohexose;dimer, trimer or other polymerized flavans;

wherein R is a C₁₋₁₀ alkyl group; and

X is a pharmaceutically acceptable counter anion of hydroxyl, chloride,iodide, sulfate, phosphate, acetate, fluoride, or carbonate.

In any of the aforementioned compositions, a Morus extract is from Morusalba, and an Acacia extract is from Acacia catechu. In furtherembodiments of these compositions, a major active ingredient in a Morusextract is Albanin G, Kuwanon G, Morusin, oxyresveratrol, mulberroside Aor any combination thereof, and a major active ingredient in an Acaciaextract is catechin, epicatechin, or both.

In further embodiments, any of the aforementioned compostions compriseone or more prenylayted flavonoids are compounds having a structure ofFormula (III), (IV) or (V), wherein the optional double bond is presentin ring C, R₁₁ and R₁₂ are absent, and R₁₀ is a prenyl group. In stillfurther embodiments, any of the aforementioned compostions comprise oneor more prenylayted flavonoids are compounds having a structure ofFormula (III), (IV) or (V), wherein the at least one of R₁-R₉ is aprenyl group and R₁₀-R₁₂ are independently H or hydroxyl. In certainembodiments, any of the aforementioned compostions comprise one or morestilbenes having a structure of Formula (I) or (II), wherein R₁-R₁₀ areeach independently a H, hydroxyl, glycoside, or C₁₋₄ alkoxy. In certainother embodiments, any of the aforementioned compostions comprise one ormore stilbenes are compounds having a structure of Formula (I) or (II),wherein R₁-R₁₀ are each independently a H, hydroxyl, glycoside, or C₁₋₄alkoxy. In further embodiments, any of the aforementioned compostionscomprise one or more stilbenes are compounds having a structure ofFormula (I) or (II), wherein R₁, R₅, R₆ and R₁₀ are H. In still furtherembodiments, any of the aforementioned compostions comprise one or morestilbenes are compounds having a structure of Formula (I) or (II),wherein R₂ is a glucoside, or R₂ and R₈ are glycosides, and one or moreof R₄, R₉, and R₁₀ are hydroxyl. In yet further embodiments, any of theaforementioned compostions comprise one or more stilbenes are compoundshaving a structure of Formula (I) or (II), wherein R₁, R₅, and R₆ are H,and one or more of R₂-R₄ and R₇-R₁₀ are independently hydroxyl, C₁₋₃alkoxy, or any combination thereof. In certain specific embodiments, astilbene compound includes oxyresveratrol, resveratrol, piceatannol,pinosylvin, 3,4′-dihydroxystilbene, combretastatin A-1, pterostilbene,rhapontigenin, and a stilbene glycoside includes mulberroside A,rhaponticin, piceid, astringin, or any combination of these stilbenes orstilbene glycosides.

Any of the aforementioned Morus extract mixed with Acacia extractcompositions are useful for promoting, managing or improving bone andcartilage health, or for preventing and treating a bone and cartilagedisorder or disease (e.g., osteoporosis, osteoarthritis, osteonecrosis,osteophyte, bone fracture, metabolic bone disorders, osteochondritisdiseases, osteochondroma, osteitis deformans, osteitis fibrosa cystica,ostteitis pubis, condensing osteitis, osteogenesis imperfecta,osteomalacia (rickets), osteomyelitis, osteopenia, or any other bone andcartilage associated indication).

In certain aspects, a composition of this disclosure comprises a mixtureof a Morus extract enriched for one or more prenylated flavonoids andone or more stilbenes, and an Acacia extract enriched for flavans,wherein the composition inhibits cartilage degradation. Cartialgedegradation is measured as the level of sulphated GAGs (e.g., releasedfrom proteoglycans) released into a medium at the end of a GAG releaseassay reaction, which reflects the amount of articular cartilagedegradation “Inhibition of cartilage degradation” is established whenthere is a statistically significant reduction in sulphated GAG releaseas measured in, for example, a Blyscan™ assay (Accurate Chemical andScientific Corp., Westbury, N.Y.) and described herein in Example 27.

In certain embodiments, a composition of this disclosure comprises anUncaria extract containing or enriched for one or more flavans asdescribed herein or in U.S. Pat. No. 8,034,387, and a Morus extractcontaining or enriched for at least one Diels-Alder adduct of a chalconeand a prenylphenyl moiety, prenylated flavonoid, stilbene, or anycombination thereof. In certain embodiments, a composition comprises anUncaria extract containing or enriched for one or more flavans asdescribed herein or in U.S. Pat. No. 8,034,387 and a Morus extractcontaining or enriched for one or more compounds listed in Table A andExamples 3, 5, 6 and 68. In still further embodiments, a compositioncomprises an Acacia extract containing or enriched for catechin,epicatichin, or both, and a Morus extract containing or enriched for oneor more prenylated flavonoids, one or more stilbenes, or any combinationthereof. In other embodiments, a composition comprises a mixture of aMorus extract enriched for prenylated flavonoids, and an Uncaria extractenriched for flavans.

In further embodiments, a composition of this disclosure comprises amixture of a Morus extract enriched for one or more prenylatedflavonoids and one or more stilbenes, and an Uncaria extract enrichedfor one or more flavans,

wherein the one or more prenylated flavonoids are compounds having astructure of Formula (III) or (IV):

or a pharmaceutically or nutraceutically acceptable salt, tautomer,glycoside, prodrug or stereoisomer thereof, wherein R₁-R₁₂ are eachindependently H, hydroxyl, a prenyl group, flavonoid, chalcone,glycoside, halogen, sulfhydryl, amino, aldehyde, C₁₋₁₂ alkyl, C₁₋₁₂alkoxy, C₁₋₁₂ alkthio, C₁₋₁₂ alkyamino, cycloalkyl, heterocyclyl, aryl,heteroaryl, aralkyl, alkyl carbonyl, aralkylcarbonyl or a bond to acompound of structure (III) or (IV); or one of R₁-R₁₂ joins with anotherone of R₁-R₁₂ to form a ring, and the remaining R₁-R₁₂ are eachindependently H, hydroxyl, a prenyl group, flavonoid, chalcone,glycoside, halogen, sulfhydryl, amino, aldehyde, C₁₋₁₂ alkyl, C₁₋₁₂alkoxy, C₁₋₁₂ alkthio, C₁₋₁₂ alkyamino, cycloalkyl, heterocyclyl, aryl,heteroaryl, aralkyl, alkylcarbonyl, aralkylcarbonyl or a bond to acompound of structure (III) or (IV), provided that all valencies aresatisfied; the chalcanoid is a compound of structure (V):

or a pharmaceutically or nutraceutically acceptable salt, tautomer,glycoside, prodrug or stereoisomer thereof, wherein R₁-R₁₀ are eachindependently H, hydroxyl, a prenyl group, flavonoid, chalcone,glycoside, halogen, sulfhydryl, amino, aldehyde, C₁₋₁₂ alkyl, C₁₋₁₂alkoxy, C₁₋₁₂ alkthio, C₁₋₁₂ alkyamino, cycloalkyl, heterocyclyl, aryl,heteroaryl, aralkyl, alkyl carbonyl, or aralkylcarbonyl, provided thatall valencies are satisfied; and the one or more stilbenes are compoundshaving a structure of Formula (I) or (II):

wherein R₁-R₁₀ are each independently a H, hydroxyl, glycoside, prenyl,flavonoid, chalcone, halogen, sulfhydryl, amino, aldehyde, C₁₋₁₂ alkyl,C₁₋₁₂ alkenyl, C₁₋₁₂ alkoxy, C₁₋₁₂ alkthio, C₁₋₁₂ alkyamino, aryl,heteroaryl, aralkyl, alkylcarbonyl, or aralkylcarbonyl; and

wherein the flavans are compounds having a structure of Formula (VI):

wherein R₂₁, R₂₂, R₂₃, R₂₄ and R₂₅ are independently selected from a H,—OH, —SH, —OCH₃, —SCH₃, —OR, —SR, —NH₂, —NRH, —NR₂, —NR₃ ⁺X⁻, esters ofsubstitution groups, independently selected from the group consisting ofgallate, acetate, cinnamoyl and hydroxyl-cinnamoyl esters,trihydroxybenzoyl esters and caffeoyl esters; a carbon, oxygen, nitrogenor sulfur glycoside of a single or a combination of multiple sugarsincluding aldopentose, methyl aldopentose, aldohexose, ketohexose;dimer, trimer or other polymerized flavans;

wherein R is a C₁₋₁₀ alkyl group; and

X is a pharmaceutically acceptable counter anion of hydroxyl, chloride,iodide, sulfate, phosphate, acetate, fluoride, or carbonate.

In any of the aforementioned compositions, the Morus extract is fromMorus alba, and the Uncaria extract is from Uncaria gambir. In furtherembodiments, a major active ingredient in the Morus extract is AlbaninG, Kuwanon G, Morusin, oxyresveratrol, mulberroside A or any combinationthereof, and a major active ingredient in the Uncaria extract iscatechin, epicatechin, or a combination thereof.

In further embodiments, the one or more prenylayted flavonoids arecompounds having a structure of Formula (III), (IV) or (V), wherein theoptional double bond is present in ring C, R₁₁ and R₁₂ are absent, andR₁₀ is a prenyl group. In still further embodiments, the one or moreprenylayted flavonoids are compounds having a structure of Formula(III), (IV) or (V), wherein the at least one of R₁-R₉ is a prenyl groupand R₁₀-R₁₂ are independently H or hydroxyl. In certain specificembodiments, the prenylated flavonoids include Albanin G, Kuwanon G,Morusin, morusinol, Sanggenon, isoxanthoumol, glabridin, cathayanon A,or any combination thereof. In certain embodiments, the one or morestilbenes are compounds having a structure of Formula (I) or (II),wherein R₁-R₁₀ are each independently a H, hydroxyl, glycoside, or C₁₋₄alkoxy. In further embodiments, the one or more stilbenes are compoundshaving a structure of Formula (I) or (II), wherein R₁, R₅, R₆ and R₁₀are H. In still further embodiments, the one or more stilbenes arecompounds having a structure of Formula (I) or (II), wherein R₂ is aglucoside, or R₂ and R₈ are glycosides, and one or more of R₄, R₉, andR₁₀ are hydroxyl. In yet further embodiments, the one or more stilbenesare compounds having a structure of Formula (I) or (II), wherein R₁, R₅,and R₆ are H, and one or more of R₂-R₄ and R₇-R₁₀ are independentlyhydroxyl, C₁₋₃ alkoxy, or any combination thereof. In certain specificembodiments, a stilbene compound includes oxyresveratrol, resveratrol,piceatannol, pinosylvin, 3,4′-dihydroxystilbene, combretastatin A-1,pterostilbene, rhapontigenin, and a stilbene glycoside includesmulberroside A, rhaponticin, piceid, astringin, or any combination ofthese stilbenes or stilbene glycosides.

In some embodiments, the flavonoid is a compound of structure (III) andin other embodiments the flavonoid is a compound of structure (IV). Insome of the embodiments, at least one of R₁-R₁₂, such as R₁₀ is prenyl.In other embodiments, polyflavonoids are provided and at least one ofR₁-R₁₂ in the compounds of structure (III) or (IV) is a bond to acompounds of structure of (III) or (IV) (i.e., the compound comprisesmore than one flavonoid of structure (III) and/or (IV)).

In some other embodiments of the compounds of structure (III) or (IV),R₁-R₁₂ is H, hydroxyl, a prenyl group or cycloalkyl. For example, insome embodiments the cycloalkyl is substituted and/or comprises one ormore carbon-carbon double bonds (i.e., is unsaturated). The optionalsubstitutents are typically selected from aryl, such as phenyl, and arylcarbonyl. Accordingly, in some further embodiments, the flavonoid hasone of the following structures (IIIa) or (IVa):

wherein R^(4a) is, at each occurrence, independently H, hydroxyl or aprenyl group.

In certain embodiments of the compounds of structure (IIIa) or (IVa),R₁-R₃ and R₅-R₁₂ are each independently selected from H, hydroxyl and aprenyl group. In certain embodiments, at least one one of R₁-R₃, R_(4a)or R₅-R₁₂ is prenyl, for example in some embodiments, R₁₀ is prenyl. Inother embodiments of the compounds of structure (IIIa) or (IVa), atleast two of R₁-R₃, R_(4a) or R₅-R₁₂ is hydroxyl.

In some more specific embodiments, the flavonoid has one of thefollowing structures:

In other embodiments, one of R₁-R₁₂ joins with another one of R₁-R₁₂ toform a ring and the remaining R₁-R₁₂ are H, hydroxyl or a prenyl group.In certain of these embodiments, the ring is a heterocyclic ring, forexample a cyclic ether ring. Accordingly, in certain embodiments theflavonoid has one of the following structures z(IIIb) or (IVb):

In certain embodiments of the compounds of structure (IIIb) or (IVb),R₁, R₂ and R₅-R₁₂ are each independently selected from H, hydroxyl and aprenyl group. In certain embodiments, at least one one of R₁, R₂ orR₅-R₁₂ is prenyl, for example in some embodiments, R₁₀ is prenyl. Inother embodiments of the compounds of structure (IIIb) or (IVb), atleast two of R₁, R₂ or R₅-R₁₂ is hydroxyl. In certain embodiments, theflavonoid has the following structure:

In various other embodiments, R₁-R₁₀ of the chalcanoid of structure (V)are each independently selected from H, hydroxyl, a prenyl group, andC₁₋₁₂ alkoxy.

Any of the aforementioned Morus extract mixed with Uncaria extractcompositions are useful for promoting, managing or improving bone andcartilage health, or for preventing and treating a bone and cartilagedisorderor disease (e.g., osteoporosis, osteoarthritis, osteonecrosis,osteophyte, bone fracture, metabolic bone disorders, osteochondritisdiseases, osteochondroma, osteitis deformans, osteitis fibrosa cystica,ostteitis pubis, condensing osteitis, osteogenesis imperfecta,osteomalacia (rickets), osteomyelitis, osteopenia, or any other bone andcartilage associated indication). In certain embodiments, a compositionof this disclosure comprises a mixture of a Morus extract enriched forone or more prenylated flavonoids and one or more stilbenes, and anUncaria extract enriched for flavans, wherein the composition inhibitsbone reabporption and cartilage degradation.

In certain embodiments, a composition comprises a mixture of a Morusextract enriched for prenylated flavonoids, an Uncaria extract enrichedfor flavans, and an Acacia extract enriched for flavans. In furtherembodiments, a composition comprises a mixture of a Morus extractenriched for one or more prenylated flavonoids and one or morestilbenes, an Uncaria extract enriched for flavans including catechin,epicatechin or both, and an Acacia extract enriched for flavansincluding catechin, epicatechin or both. In certain embodiments, theMorus extract is from Morus alba, the Uncaria extract is from Uncariagambir, and the Acacia extract is from Acacia catechu. In furtherembodiments, a major active ingredient in the Morus extract is AlbaninG, Kuwanon G, Morusin, oxyresveratrol, mulberroside A or any combinationthereof, and a major active ingredient in the Uncaria and Acaciaextracts is catechin, epicatechin, or a combination thereof. Any ofthese three extract compositions (Morus, Uncaria, Acacia) are useful forpromoting, managing or improving joint health, or for preventing andtreating a bone and cartilage disorderor disease (e.g., osteoporosis,osteoarthritis, osteonecrosis, osteophyte, bone fracture, metabolic bonedisorders, osteochondritis diseases, osteochondroma, osteitis deformans,osteitis fibrosa cystica, ostteitis pubis, condensing osteitis,osteogenesis imperfecta, osteomalacia (rickets), osteomyelitis,osteopenia, or any other bone and cartilage associated indication).

In certain embodiments, a composition of this disclosure comprises amixture of a Morus extract containing or enriched for at least oneDiels-Alder adduct of a chalcone and a prenylphenyl moiety, prenylatedflavonoid, stilbene or any combination thereof, and a Curcuma extractenriched for curcuminoids. In further embodiments, a compositioncomprises a mixture of a Morus extract containing or enriched for one ormore compounds listed in Table A and Examples 3, 5, 6 and 68, and aCurcuma extract enriched for one or more curcuminoids. In still furtherembodiments, a composition comprises a Morus extract containing orenriched for one or more prenylated flavonoids, one or more stilbenes orany combination thereof, and a Curcuma extract enriched for one or morecurcuminoids. In certain embodiments, the Morus extract is from Morusalba, and the Curcuma extract is from Curcuma longa. In any of theaforementioned compositions, a major active ingredient in the Morusextract is Albanin G, Kuwanon G, Morusin, oxyresveratrol, mulberroside Aor any combination thereof, and a major active ingredient in the Curcumaextract is curcumin, demethoxy-curcumin, bisdemethoxy-curcumin or anycombination thereof.

Any of the aforementioned Morus extract mixed with Curcuma extractcompositions are useful for promoting, managing or improving bone andcartilage health, or for preventing, or treating a bone and cartilagedisorderor disease (e.g., osteoporosis, osteoarthritis, osteonecrosis,osteophyte, bone fracture, metabolic bone disorders, osteochondritisdiseases, osteochondroma, osteitis deformans, osteitis fibrosa cystica,ostteitis pubis, condensing osteitis, osteogenesis imperfecta,osteomalacia (rickets), osteomyelitis, osteopenia, or any other bone andcartilage associated indication). In certain embodiments, a compositionof this disclosure comprises a mixture of a Morus extract enriched forone or more prenylated flavonoids and one or more stilbenes, and anCurcuma extract enriched for one or more curcuminoids, wherein thecomposition inhibits bone reabsorption and cartilage degradation.

In certain embodiments, a composition comprises a mixture of a Morusextract enriched for prenylated flavonoids, an Acacia extract enrichedfor flavans, and a Curcuma extract enriched for curcuminoids. In furtherembodiments, a composition comprises a mixture of a Morus extractenriched for one or more prenylated flavonoids and one or morestilbenes, an Acacia extract enriched for flavans including catechin,epicatechin or both, and a Curcuma extract enriched for one or morecurcuminoids. In certain embodiments, the Morus extract is from Morusalba, the Acacia extract is from Acacia catechu, and the Curcuma extractis from Curcuma longa. In further embodiments, a major active ingredientin the Morus extract is Albanin G, Kuwanon G, Morusin, oxyresveratrol,mulberroside A or any combination thereof, and a major active ingredientin the Curcuma extract is curcumin (diferuloylmethane),demethoxy-curcumin, bisdemethoxy-curcumin or any combination thereof.

In certain embodiments, a composition comprises a mixture of a Morusextract enriched for prenylated flavonoids, an Uncaria extract enrichedfor flavans, and a Curcuma extract enriched for curcuminoids. In furtherembodiments, a composition comprises a mixture of a Morus extractenriched for one or more prenylated flavonoids and one or morestilbenes, an Uncaria extract enriched for flavans including catechin,epicatechin or both, and a Curcuma extract enriched for one or morecurcuminoids. In certain embodiments, the Morus extract is from Morusalba, the Uncaria extract is from Uncaria gambir, and the Curcumaextract is from Curcuma longa.

Any of these three extract compositions (Morus, Morus, Acacia, Curcumaor Morus, Uncaria, Curcuma) are useful for promoting, managing orimproving bone health, cartilage health or both, or for preventing orfor treating a bone disorder, a cartilage disorder or both (e.g.,osteoporosis, osteoarthritis, osteonecrosis, osteophyte, bone fracture,metabolic bone disorders, osteochondritis diseases, osteochondroma,osteitis deformans, osteitis fibrosa cystica, ostteitis pubis,condensing osteitis, osteogenesis imperfecta, osteomalacia (rickets),osteomyelitis, osteopenia, or any other bone or cartilage associatedindication).

In any of the aforementioned compositions, a Morus extract is enrichedfor prenylated flavonoids, such as Albanin G, Kuwanon G, Morusin, or anycombination thereof. In certain embodiments, a Morus extract is enrichedfor stilbenes, such as oxyresveratrol, mulberroside A, or anycombination thereof. In further embodiments, a Morus extract is enrichedfor prenylated flavonoids and stilbenes, including Albanin G, Kuwanon G,Morusin, oxyresveratrol, mulberroside A, or any combination thereof. Instill further embodiments, a Morus extract is enriched for prenylatedflavonoids and stilbenes, wherein the extract comprises from about 2% toabout 25% prenylated flavonoids and from about 1% to about 8% stilbenes,or wherein the extract comprises at least 3% prenylated flavonoids andat least 3% stilbenes (weight to weight). In other embodiments,prenylated flavonoids, stilbenes, or both are isolated or purified froma Morus extract and used in the compositions of this disclosure.Exemplary active ingredients that can be isolated or purified from aMorus extract and used in the compositions of this disclosure includeAlbanin G, Kuwanon G, Morusin, oxyresveratrol, mulberroside A, or anycombination thereof. In any of the aforementioned compositions, theMorus extract is from Morus alba.

In any of the aforementioned embodiments, the compositions comprisingmixtures of extracts or compounds may be mixed at a particular ratio byweight. For example, a Morus extract and an Acacia extract may beblended in a 2:1 weight ratio, respectively. In certain embodiments, theratio (by weight) of two extracts or compounds of this disclosure rangesfrom about 0.5:5 to about 5:0.5. Similar ranges apply when more than twoextracts or compounds (e.g., three, four, five) are used. Exemplaryratios include 0.5:1, 0.5:2, 0.5:3, 0.5:4, 0.5:5, 1:1, 1:2, 1:3, 1:4,1:5, 2:1, 2:2, 2:3, 2:4, 2:5, 3:1, 3:2, 3:3, 3:4, 3:5, 4:1, 4:2, 4:3,4:4, 4:5, 5:1, 5:2, 5:3, 5:4, 5:5, 1:0.5, 2:0.5, 3:0.5, 4:0.5, or 5:0.5.In certain embodiments, Morus and Acacia extracts are blended in a 1:1,2:1, 3:1, 4:1, 5:1, 1:2, 1:3, 1:4, or 1:5 weight ratio, respectively. Infurther embodiments, Morus and Acacia extracts are blended in a range of1:2 to 4:1 weight ratio, respectively. In certain embodiments, Morus andUncaria extracts are blended in a 1:1, 2:1, 3:1, 4:1, 5:1, 1:2, 1:3,1:4, or 1:5 weight ratio, respectively. In further embodiments, Morusand Uncaria extracts are blended in a range of 1:4 to 4:1 weight ratio,respectively. In certain embodiments, Morus and Curcuma extracts areblended in a 1:1, 2:1, 3:1, 4:1, 5:1, 1:2, 1:3, 1:4, or 1:5 weightratio, respectively. In further embodiments, Morus and Curcuma extractsare blended in a range of 1:1 to 4:1 weight ratio, respectively.

In any of the aforementioned embodiments, the compositions comprisingmixtures of extracts or compounds may be present at certain percentagelevels or ratios. In certain embodiments, a composition comprising aMorus extract can include 0.1% to 49.9% or about 1% to about 10% orabout 0.5% to about 3% of prenylated flavonoids, 0.1% to 49.9% or about1% to about 10% or about 0.5% to about 3% of stilbenes, or a combinationthereof. In certain embodiments, a composition comprising an Acaciaextract can include from about 0.01% to about 99.9% flavans or includeat least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%,15%, 16%, 17%, 18%, 19%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, or 80% flavans (e.g., catechin, epicatechin, or both)

In certain examples, a composition of this disclosure may be formulatedto further comprise a pharmaceutically or nutraceutically acceptablecarrier, diluent, or excipient, wherein the pharmaceutical ornutraceutical formulation comprises from about 0.5 weight percent (wt %)to about 90 wt % of active or major active ingredients of an extractmixture. In further embodiments, the pharmaceutical or nutraceuticalformulation comprises from about 0.5 weight percent (wt %) to about 90wt %, about 0.5 wt % to about 80 wt %, about 0.5 wt % to about 75 wt %,about 0.5 wt % to about 70 wt %, about 0.5 wt % to about 50 wt %, about1.0 wt % to about 40 wt %, about 1.0 wt % to about 20 wt %, about 1.0 wt% to about 10 wt %, about 3.0 wt % to about 9.0 wt %, about 5.0 wt % toabout 10 wt %, about 3.0 wt % to about 6 wt % of the major activeingredients in an extract mixture, or the like. In any of theaforementioned formulations, a composition of this disclosure isformulated as a tablet, hard capsule, softgel capsule, powder, orgranule.

In certain embodiments, a composition comprising a Morus extract with apharmaceutically or nutraceutically acceptable carrier, diluent, orexcipient will contain at least 6 wt % or at least 5 wt % or at least 3wt % or at least 2 wt % or at least 1 wt % active Morus ingredients,such as prenylated flavonoids, stilbenes, or a combination thereof. Forexample, a pharmaceutical or nutraceutical composition comprising aMorus extract will include at least 3 wt % prenylated flavonoids or fromabout at least 0.5 wt % to about at least 2.5 wt % or from about atleast 1 wt % to about at least 2.5 wt % or from about at least 1.5 wt %to about at least 2.5 wt % (e.g., Albanin G, Kuwanon G, Morusin, or anycombination thereof) and at least 3% stilbenes (e.g., oxyresveratrol,mulberroside A, or both). In certain embodiments, a compositioncomprising an Acacia or Uncaria extract with a pharmaceutically ornutraceutically acceptable carrier, diluent, or excipient will containat least 20 wt % active Acacia or Uncaria ingredients, such as flavans.For example, a pharmaceutical or nutraceutical composition comprising anAcacia or Uncaria extract will include at least about 3.5 wt % to aboutat least 14 wt % or at least about 6 wt % to about at least 16.5 wt %(e.g., catechin, epicatechin, or both). In certain embodiments, acomposition comprising a Curcuma extract with a pharmaceutically ornutraceutically acceptable carrier, diluent, or excipient will containat least 25 wt % active Curcuma ingredients, such as cucuminoids. Forexample, a pharmaceutical or nutraceutical composition comprising aCurcuma extract will include at least about 4.5 wt % to at least about13 wt % curcuminoids (e.g., curcumin, demethoxy-curcumin,bisdemethoxy-curcumin, or any combination thereof). In any of theaforementioned formulations, a composition of this disclosure isformulated as a tablet, hard capsule, softgel capsule, powder, orgranule.

In certain embodiments, a composition of this disclosure comprises Morusand Acacia extracts, wherein the composition comprises from about 1 wt %to about 2.5 wt % prenylated flavonoids including Albanin G, Kuwanon Gand Morusin, from about 1 wt % to about 2.5 wt % stilbenes includingoxyresveratrol and mulberroside A, and about 3.5 wt % to about 14 wt %flavans including catechin and epicatechin. In certain otherembodiments, a composition of this disclosure comprises Morus andUncaria extracts, wherein the composition comprises from about 0.5 wt %to about 2.5 wt % prenylated flavonoids including Albanin G, Kuwanon Gand Morusin, from about 0.5 wt % to about 2.5 wt % stilbenes includingoxyresveratrol and mulberroside A, and about 6 wt % to about 16.5 wt %flavans including catechin and epicatechin. In certain furtherembodiments, a composition of this disclosure comprises Morus andCurcuma extracts, wherein the composition comprises from about 1.5 wt %to about 2.5 wt % prenylated flavonoids including Albanin G, Kuwanon Gand Morusin, from about 1.5 wt % to about 2.5 wt % stilbenes includingoxyresveratrol and mulberroside A, and about 4.5 wt % to about 13 wt %curcuminoids including curcumin.

Any of these compositions may be used to promote joint health; improvejoint health; maintain joint health; treat or manage joint health;support joint health; support a normal and comfortable range of motionand/or flexibility; improve range of motion and/or flexibility; reducethe action of harmful enzymes that break down protective joint tissues;alter the action of enzymes that affect joint health; improve jointmovement and/or joint function; improve physical mobility; manage and/ormaintain physical mobility; alleviate joint pain and/or joint stiffness;improve joint physical function; promote or enhance flexibility andcomfortable movement; promote healthy joint function and joint comfort;relieve joint discomfort; relieve joint discomfort caused by exercise,work, overexertion or any combination thereof; promote healthy joints byprotecting cartilage integrity; maintain joint cartilage; support jointcartilage; treat, prevent, or manage cartilage degradation; minimizecartilage degradation; promote joint health or comfort by maintainingsynovial fluid for joint lubrication; support joint stability and jointflexibility; revitalize joints and promote mobility; promote flexiblejoints and strong cartilage; maintain steady blood flow to joints tosupport enhanced flexibility and/or strength; promote joint comfort anda wide range of motion after exercise, work, overexertion, or anycombination thereof.

In other embodiments, any of these compositions may be useful forpromoting, managing or improving bone health, cartilage health or both,or for preventing or treating a bone disorder, a cartilage disorder(e.g., osteoporosis, osteoarthritis, osteonecrosis, osteophyte, bonefracture, metabolic bone disorders, osteochondritis diseases,osteochondroma, osteitis deformans, osteitis fibrosa cystica, ostteitispubis, condensing osteitis, osteogenesis imperfecta, osteomalacia(rickets), osteomyelitis, osteopenia, or any other bone and cartilageassociated indication).

In other embodiments of the present disclosure, a composition can alsoinclude an adjuvant or a carrier. Adjuvants include substances thatgenerally enhance the function of the formula in promoting, maintaining,or improving joint health. Suitable adjuvants include Freund's adjuvant;other bacterial cell wall components; aluminum-based salts;calcium-based salts; magnesium, zinc, silica; boron, histidine,glucosamine sulfates, Chondroitin sulfate, copper gluconate,polynucleotides; vitamin D, vitamin K, toxoids; shark and bovinecartilage; serum proteins; viral coat proteins; other bacterial-derivedpreparations; γ-interferon; block copolymer adjuvants, such as Hunter'sTitermax adjuvant (Vaxcel™, Inc. Norcross, Ga.); Ribi adjuvants(available from Ribi ImmunoChem Research, Inc., Hamilton, Mont.); andsaponins and their derivatives, such as Quil A (available from SuperfosBiosector A/S, Denmark). Carriers include compounds that increase thehalf-life of a therapeutic or neutraceutical composition in a treatedsubject. Suitable carriers include polymeric controlled releaseformulations, biodegradable implants, liposomes, bacteria, viruses,oils, esters, or glycols.

Additional adjunctive agents useful with the compositions of thisdisclosure include glucosamine (including glucosamine sulfate,glucosamine hydrochloride, N-acetylglucosamine), glycosaminoglycans(GAGs), hyaluronic acid (HA), elastin, collagen, chicken collagen TypeII, hyaluronic acid and collagen blend, chondroitin sulfate,methylsulfonylmethane (MSM), bovine cartilage, amino acids (includingdesmosine, isodesmosine, L-glutamine), Boswellia serrata extract,piperine (e.g., Piper nigrum L (black pepper) extract or Piper longum L(long pepper) extract), bromelain (pineapple extract), trypsin, rutin,emu oil, transforming growth factor(TGF)-β, carotenoids (such as lutein,carotene, canthaxanthin); vitamins (such as Vitamin D3), ω-3 fatty acids(such as eicosapentaenoic acid, EPA; docosahexaenoic acid, DHA), calciumfructoborate, eggshell membrane, astaxanthin, Hydrilla verticillataextract (leaf and bud), ginger extract (root), grapefruit extract(seed), non-steroidal anti-inflammatory drugs (NSAIDs), or anycombination thereof.

Exemplary NSAIDS include salicylates, such as aspirin (acetylsalicylicacid), diflusinal, salsalate; propionic acid derivatives, such asibuprofen, dexibuprofen, naproxen, fenoprofen, ketoprofen,dexketoprofen, flurbiprofen, oxprozin, loxoprofen; acetic acidderivatives, such as indometacin, tolmetin, sulindac, etodolac,ketorolac, diclofenac, nabumetone; enolic acid derivatives, such aspiroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, isoxicam; fenamicacid derivatives, such as mefenamic acid, meclofenamic acid, flufenamicacid, tolfenamic acid; selective COX-2 inhibitors, such s celecoxib,parecoxib, lumiracoxib, etoricoxib, firocoxib, paracetamol, H-harpagide;suphonanilides, such as nimesulide; nicotinic acid derivatives, such aslysine clonixinate; dual COX/LX inhibitors, such as licofelone. Arelated drug, paracetamol or “acetaminophen” is often considered in thesame category as NSAIDS due to its use as a non-narcotic analgesic andfever-reducing agent, but is not classified as a NSAID because it onlyexerts weak anti-inflammatory activity.

In certain embodiments, compositions of the instant disclosure furthercomprise an injectable anticoagulant, an oral anticoagulant, anantiplatelet agent, an anti-angina agent, or a COX-2 selectiveinhibitor. Exemplary injectable anticoagulants include heparin,dalteparin, enoxaparin and tinzaparin. Examples of oral anticoagulantsinclude, but are not limited to warfarin, vitamin K antagonists andvitamin K reductase inhibitors. Examples of antiplatelet agents includeaspirin, clodipogrel and dipyridamole. Examplary anti-angina drugsinclude nitrates, beta-blockers, calcium blockers,angiotensin-converting enzyme inhibitors, and potassium channelactivators. Finally, examples of COX-2 selective inhibitors includerofecoxib, celecoxib, etodolac and meloxicam.

In certain embodiments, a composition comprises a mixture of a Morusextract enriched for one or more prenylated flavonoids and one or morestilbenes, an Acacia extract enriched for flavans, and aglucosamine-type compound. In further embodiments, the Morus extract isa Morus alba extract, the Acacia extract is an Acacia catechu extract,and the glucosamine-type compound is glucosamine sulfate, glucosaminehydrochloride, N-acetylglucosamine, chondroitin sulfate,methylsulfonylmethane, and/or hyaluronic acid. In certain embodiments,Morus extract, Acacia extract, and NAG are blended in a 1:1:1, 2:1:1,3:1:1, 4:1:1, 5:1:1, 1:2:1, 1:3:1, 1:4:1, 1:5:1, 1:1:2, 1:1:3, 1:1:4, or1:1:5 weight ratio, respectively. In certain embodiments, Morus extract,Uncaria extract, and NAG are blended in a 1:1:1, 2:1:1, 3:1:1, 4:1:1,5:1:1, 1:2:1, 1:3:1, 1:4:1, 1:5:1, 1:1:2, 1:1:3, 1:1:4, or 1:1:5 weightratio, respectively. In certain embodiments, Morus extract, Curcumaextract, and NAG are blended in a 1:1:1, 2:1:1, 3:1:1, 4:1:1, 5:1:1,1:2:1, 1:3:1, 1:4:1, 1:5:1, 1:1:2, 1:1:3, 1:1:4, or 1:1:5 weight ratio,respectively. In certain embodiments, a composition comprises a mixtureof a Morus extract enriched for prenylated flavonoids, an Uncariaextract enriched for flavans, and a glucosamine-type compound. Infurther embodiments, the Morus extract is a Morus alba extract, theUncaria extract is an Uncaria gambir extract, and the glucosamine-typecompound is glucosamine sulfate, glucosamine hydrochloride,N-acetylglucosamine, chondroitin sulfate, methylsulfonylmethane, orhyaluronic acid.

In certain embodiments, a composition comprises a mixture of a Morusextract enriched for prenylated flavonoids, a Curcuma extract enrichedfor curcuminoids, and a glucosamine-type compound. In furtherembodiments, the Morus extract is a Morus alba extract, the Curcumaextract is a Curcuma longa extract, and the glucosamine-type compound isglucosamine sulfate, glucosamine hydrochloride, N-acetylglucosamine,chondroitin sulfate, methylsulfonylmethane, or hyaluronic acid.

In any of the aforementioned compositions, the compositions mayadditionally comprise Mentha extract enriched for rosmarinic acid,eriocitrin, or both. Rosmarinic acid accumulation is found most notablyin many plants of the Lamiaceae family (dicotyledons), especially in thesubfamily Nepetoideae, inlcuding plants commonly used as culinary herbs,such as Ocimum basilicum (basil), Ocimum tenuiflorumcum (holy basil),Melissa officinalis (lemon balm), Rosmarinus officinalis (rosemary),Origanum majorana (marjoram), Salvia officinalis (sage), Thymus vulgaris(thyme) and Mentha piperita (peppermint). Rosmarinic acid is also foundin plants with medicinal properties, such as common self-heal (Prunellavulgaris) or species in the genus Stachy. Other exemplary plants thatcontain rosmarinic acid include Heliotropium foertherianum (a plant inthe family Boraginaceae), species in the genera Maranta (Marantaleuconeura, Maranta depressa, which are plants in the familyMarantaceae, monocotyledons), species in the genera Thalia (Thaliageniculata), and Anthoceros agrestis (hornwort).

Exemplary mint plants containing rosmarinic acid or eriocitrin or bothinclude Mentha aquatica (Water mint or Marsh mint); Mentha arvensis(Corn Mint, Wild Mint, Japanese Peppermint, Field Mint, Pudina, Bananamint); Mentha asiatica (Asian Mint); Mentha australis (Australian mint);Mentha canadensis; Mentha cervina (Hart's Pennyroyal); Mentha citrata(Bergamot mint, Orange mint); Mentha crispata (Wrinkled-leaf mint);Mentha dahurica (Dahurian Thyme); Mentha diemenica (Slender mint);Mentha laxiflora (Forest mint); Mentha longifolia (Mentha sylvestris,Horse Mint); Mentha piperita (Peppermint); Mentha pulegium (Pennyroyal);Mentha requienii (Corsican mint); Mentha sachalinensis (Garden mint);Mentha satureioides (Native Pennyroyal); Mentha spicata (M. viridis, synM. cordifolia Spearmint, Curly mint); Mentha suaveolens (Apple mint,Pineapple mint (a variegated cultivar of Apple mint)); Mentha vagans(Gray mint).

In certain embodiments, a composition comprises a mixture of a Morusextract enriched for prenylated flavonoids, an Acacia extract enrichedfor flavans, and a Mentha extract enriched for rosmarinic acid,eriocitrin, or both. In further embodiments, the Morus extract is aMorus alba extract, the Acacia extract is an Acacia catechu extract, andthe Mentha extract is a Mentha piperita extract. In certain embodiments,Morus, Acacia and Mentha extracts are blended in a 1:1:0.5, 2:1:0.5,3:1:0.5, 4:1:0.5, 5:1:0.5, 1:2:0.5, 1:3:0.5, 1:4:0.5, 1:5:0.5, 1:1:1,1:1:2, 1:1:3, 1:1:4, or 1:1:5 weight ratio, respectively.

In certain embodiments, a composition comprises a mixture of a Morusextract enriched for prenylated flavonoids, an Uncaria extract enrichedfor flavans, and a Mentha extract enriched for rosmarinic acid,eriocitrin, or both. In further embodiments, the Morus extract is aMorus alba extract, the Uncaria extract is an Uncaria gambir extract,and the Mentha extract is a Mentha piperita extract. In certainembodiments, Morus, Uncaria and Mentha extracts are blended in a1:1:0.5, 2:1:0.5, 3:1:0.5, 4:1:0.5, 5:1:0.5, 1:2:0.5, 1:3:0.5, 1:4:0.5,1:5:0.5, 1:1:1, 1:1:2, 1:1:3, 1:1:4, or 1:1:5 weight ratio,respectively.

In certain embodiments, a composition comprises a mixture of a Morusextract enriched for prenylated flavonoids, a Curcuma extract enrichedfor curcuminoids, and a Mentha extract enriched for rosmarinic acid,eriocitrin, or both. In further embodiments, the Morus extract is aMorus alba extract, the Curcuma extract is a Curcuma longa extract, andthe Mentha extract is a Mentha piperita extract. In certain embodiments,Morus, Curcuma and Mentha extracts are blended in a 1:1:0.5, 2:1:0.5,3:1:0.5, 4:1:0.5, 5:1:0.5, 1:2:0.5, 1:3:0.5, 1:4:0.5, 1:5:0.5, 1:1:1,1:1:2, 1:1:3, 1:1:4, or 1:1:5 weight ratio, respectively.

Any of the aforementioned compositions are useful for promoting bonehealth, cartilage health or both; improving bone health, cartilagehealth or both; maintaining bone health, cartilage health or both;treating or managing bone health, cartilage health or both; supportingbone health, cartilage health or both; supporting a normal andcomfortable range of motion or flexibility; improving range of motion orflexibility; reducing the action of harmful enzymes that break downprotective bone tissue, cartilage tissue or both; altering the action ofenzymes that affect bone health, cartilage health or both; improvingjoint movement or bone function, cartilage function or both; improvingphysical mobility; managing or maintaining physical mobility;alleviating bone and cartilage pain or joint stiffness; improvingphysical function of bone or cartilage; promoting or enhancingflexibility and comfortable movement; promoting healthy bone function,cartilage function, joint comfort or any combination thereof; relievingdiscomfort; relieving discomfort caused by oxidative stress, harmfulfree radicals, aging, wear and tear, exercise, work, overexertion or anycombination thereof; managing or reducing bone damage, cartilage damageor both caused by oxidative stress, harmful free radicals, aging, wearand tear, exercise, work, overexertion or any combination thereof;promoting healthy bone, healthy cartilage or both by protecting boneintegrity, cartilage integrity or both; maintaining bone, cartilage orboth; supporting bone, supporting cartilage or both; treating,preventing, or managing bone absorption, cartilage degradation or both;minimizing cartilage degradation; promoting bone health, cartilagehealth, joint comfort, or any combination thereof by maintainingsynovial fluid for joint lubrication; supporting bone stability;revitalizing bone, cartilage or both to promote mobility; promotingflexible joints, strong cartilage or both; maintaining steady blood flowto bone to support enhanced bone strength; promoting comfort and a widerange of motion after exercise, work, overexertion or any combinationthereof.

In further embodiments, any of the aforementioned compositions areuseful for promoting, managing or improving bone health, cartilagehealth or both, or for preventing, managing or treating a bone disorder,cartilage disorder or both (such as osteoporosis, osteoarthritis,osteonecrosis, osteophyte, bone fracture, metabolic bone disorders,osteochondritis diseases, osteochondroma, osteitis deformans, osteitisfibrosa cystica, ostteitis pubis, condensing osteitis, osteogenesisimperfecta, osteomalacia (rickets), osteomyelitis, osteopenia), or anyother bone- or cartilage-associated indication, or any combinationthereof.

Bone is constantly undergoing a metabolic process called remodeling.This includes a degradation process, bone reabsorption, and a buildingprocess, bone formation. Cross-linked telopeptides collagens are theproducts in the remodeling process. While telopeptide of type I collagenaccounts about 90% of the organic matrix of bone, the type II collagenis the major organic constituent of cartilage. Disruption of thestructural integrity of cartilage is the major histological finding inosteoarthritis and rheumatoid arthritis. Following the degradation ofcartilage, fragments of C-terminal cross-linked telopeptide type IIcollagen (CTX-II) are being released into circulation and subsequentlysecreted into urine. Therefore, (CTX-II) is considered a viablebiomarker for cartilage degradation and disease progression. In multiplestudies, urinary CTX-II has been reported to be useful indicator inprogression of osteoarthritis, and early indication of rheumatoidarthritis.

As osteoarthritis progresses, the joint components including matrix andcartilage are degraded by proteases. The degraded products such asCTX-II are released into the serum and urine, and the CTX-IIconcentration in body fluids reflects OA progression. Levels of CTX-IIcan be measured by known assays, such as the one described in Example 38herein.

In certain embodiments, the promotion, management or improvement of bonehealth, cartilage health or both, or prevention, management or treatmentof a bone disorder, cartilage disorder or both (such as osteoporosis,osteoarthritis, osteonecrosis, osteophyte, bone fracture, metabolic bonedisorders, osteochondritis diseases, osteochondroma, osteitis deformans,osteitis fibrosa cystica, ostteitis pubis, condensing osteitis,osteogenesis imperfecta, osteomalacia (rickets), osteomyelitis,osteopenia), or any other bone- or cartilage-associated indication, orany combination thereof, is detected by measuring a biomarker, such asCTX-II.

EXAMPLES Example 1 Preparation of Organic and Aqueous Extracts fromMorus alba

Plant material from Morus alba L. root barks was ground to a particlesize of no larger than two millimeters (mm). Dried ground plant material(60 grams (g) was then transferred to an Erlenmeyer flask andMethanol:Dichloromethane (1:1 volume ratio) (600 milliliters (mL)) wasadded. The mixture was shaken for one hour, filtered and the biomass wasextracted again with Methanol:Dichloromethane (1:1 volume ratio) (600mL). These organic extracts were combined and evaporated under vacuum toprovide 3.55 g of organic extract (OE). After organic extraction, thebiomass was air dried and extracted once with ultrapure water (600 mL).The aqueous solution was filtered and freeze-dried to provide 4.44 g ofaqueous extract (AE).

Similar results were obtained using the same procedure or reflex inflasks, but with the organic solvent being replaced with methanol orethanol to provide a methanol extract (ME) or ethanol extract (EE),respectively. Other species and parts of plants and marine sample wereextracted using this same procedure.

Example 2 High Throughput Purification (HTP) of Active Plant Extracts

Organic extract material (400 mg) from the Morus alba root bark extractobtained in Example 1 was loaded onto a prepacked (2 cm ID×8.2 cm, 10 gsilica gel) column. The column was then eluted using a Hitachi® HighThroughput Purification (HTP) system with a gradient mobile phase of (A)50:50 volume ratio of EtOAc:Hexane and (B) Methanol from 100% A to 100%B in 30 minutes at a flow rate of 5 mL/min. The separation was monitoredusing a broadband wavelength UV detector and the fractions werecollected in a 96-deep-well plate at 1.9 mL/well using a Gilson fractioncollector. The sample plate was dried under low vacuum andcentrifugation and then the samples were dissolved with 1.5 mL dimethylsulfoxide (DMSO) per well. A portion (100 μL) was taken and combined(based on UV trace) for the function assay. Column fractions havingsignificant biological activity were retained for further testing.

Example 3 Isolation, Purification, and Identification of PrenylatedFlavonoids from Morus alba Extracts

An organic extract (11 g) from the root barks of Morus alba, obtained asdescribed in Example 1, was divided and loaded separately onto twopre-packed flash columns (120 g silica, particle size 32-60 μm, 4 cm×19cm), and then eluted with Hexane, EtOAc and Methanol (as the mobilephase) at a flow rate of 20 mL/minutes. The gradients started with 95%Hexane/EtOAC for 5 minutes, then increased EtOAC from 5% to 100% overthe duration of 25 minutes, and then held at 100% EtOAc for additionalfive minutes, before increasing MeOH from 0% to 50% MeOH/EtOAC over anext period of 15 minutes, finally changed the elution solution to 100%MeOH and eluted the column for another 16 minutes. The total run timewas 66 minutes and 88 fractions were generated for each column. Thefractions were analyzed by silica gel thin layer chromatography (TLC)and pooled together to generate eight column eluent pools.

The resulting best active pool (containing 300 mg of material) wasfractionated on a preparative C18 column (30 cm×250 cm) with a gradientmobile phase of water (A) and methanol (B) over 60 minutes at a flowrate of 20 mL/minute to generate 22 fraction pools. Mass Spectrometry(MS) analysis showed that these pooled fractions of material containthree related compounds, described in more detail below.

Compound 1 (28.2 mg) was identified as a Diels-Alder adduct of achalcone and prenylphenyl moiety called Kuwanon G, also known asMoracenin B or Albanin F, by High Resolution Electron Spray IonizationMass Spectroscopy (HRESIMS) (m/z) [M+H]⁻=693.2329; UV λ_(max) (MeOH):265, 320 nm; ¹H NMR (600 MHz, DMSO-d₆, 100° C.) δ ppm 1.44 (s, 3H) 1.52(br. s., 3H) 1.58 (s, 3H) 1.92 (m, 2H) 3.08 (d, 3H) 3.56 (m, 2H) 4.29(d, J=10.02 Hz, 1H) 4.48 (m, 1H) 5.07 (m, 1H) 5.14 (br. s, 1H) 5.93 (s,2H) 5.96 (dd, J=8.35, 2.23 Hz, 1H) 6.02 (br s, 1H) 6.11 (d, J=2.23 Hz,1H) 6.41 (dd, J=8.35, 2.23 Hz, 1H) 6.51 (s, 1H) 6.60 (m, 1H) 7.13 (d,J=8.35 Hz, 1H) 7.28 (br s, 1H); ¹³C NMR (126 MHz, METHANOL-d₄) δ ppm16.35 (1C) 21.78 (1C) 23.35 (1C) 24.53 (1C) 37.72 (1C) 97.14 (1C) 101.57(1C) 102.22 (1C) 102.33 (1C) 104.28 (1C) 106.55 (2C) 107.00 (1C) 107.21(1C) 112.37 (1C) 114.47 (1C) 120.27 (1C) 121.62 (2C) 123.27 (1C) 131.05(1C) 131.35 (2C) 132.62 (1C) 132.99 (1C) 155.16 (1C) 155.56 (1C) 156.38(1C) 159.66 (1C) 160.39 (2C) 161.13 (1C) 161.88 (1C) 164.51 (1C) 164.63(1C) 182.46 (1C) 208.68 (1C).

Compound 2 (10.5 mg) was identified as Albanin G, also known as KuwanonH or Moracenin A, another Diels-Alder adduct of a chalcone andprenylphenyl moiety by HRESIMS (m/z) [M−H]⁻=759; UV λ_(max) (MeOH): 265,320 nm; ¹³C NMR (126 MHz, METHANOL-d₄) δ ppm 16.35 (1C) 16.47 (1C) 20.96(1C) 21.79 (1C) 23.32 (1C) 24.51 (1C) 24.53 (1C) 33.74 (1C) 35.61 (1C)36.81 (1C) 37.77 (1C) 97.19 (1C) 102.27 (1C) 102.33 (1C) 104.24 (1C)106.07 (1C) 106.53 (2C) 107.34 (1C) 112.37 (1C) 113.94 (1C) 114.35 (1C)120.17 (1C) 121.60 (2C) 122.31 (2C) 123.25 (1C) 130.21 (2C) 131.33 (2C)132.96 (1C) 156.37 (3C) 157.07 (1C) 159.59 (1C) 160.37 (1C) 161.23 (1C)161.77 (1C) 161.96 (1C) 162.21 (1C) 182.45 (1C) 208.82 (1C).

Compound 3 (12.9 mg) was identified as Morusinol by ESIMS (m/z)[M−H]⁻=437; UV λ_(max) (MeOH): 269, 317 nm; ¹H NMR (500 MHz,METHANOL-d₄) δ ppm 1.08 (s, 6H) 1.43 (s, 6H) 1.60 (m, 2H) 2.43 (m, 2H)5.59 (d, J=9.97 Hz, 1H) 6.16 (s, 1H) 6.43 (m, 2H) 6.59 (d, J=10.26 Hz,1H) 7.15 (d, J=9.09 Hz, 1H); ¹³C NMR (126 MHz, METHANOL-d₄) δ ppm 21.52(t, 1C) 28.54 (q, 2C) 28.88 (q, 2C) 43.19 (t, 1C) 71.56 (s, 1C) 79.28(s, 1C) 100.28 (d, 1C) 102.35 (s, 1C) 104.06 (d, 1C) 106.05 (s, 1C)108.26 (d, 1C) 113.14 (s, 1C) 115.89 (d, 1C) 122.99 (s, 1C) 128.36 (d,1C) 132.37 (d, 1C) 153.97 (s, 1C) 157.96 (s, 1C) 160.62 (s, 1C) 162.13(s, 1C) 162.88 (s, 1C) 163.63 (s, 1C) 184.09 (s, 1C)

Another best active pool (containing 538 mg of material) wasfractionated on a preparative C18 column (30 cm×250 cm) with a gradientmobile phase of water (A) and methanol (B) over 60 minutes at a flowrate of 20 mL/minute to generate 16 fraction pools. A prenylphenylatedCompound 4, called Morusin (80 mg), also known as Mulberrochromene wasisolated. The structure and spectroscopy data were as follows: ESIMS(m/z) [M−H]⁻ 419; UV λmax (MeOH): 269.4 nm; 1H NMR (500 MHz,METHANOL-d4) δ ppm 1.41 (m, 9H) 1.58 (s, 3H) 3.10 (d, J=7.15 Hz, 2H)5.09 (m, 1H) 5.57 (d, J=10.49 Hz, 1H) 6.14 (s, 1H) 6.40 (m, 2H) 6.59 (d,J=10.01 Hz, 1H) 7.10 (d, J=8.11 Hz, 1H); 13C NMR (126 MHz, METHANOL-d4)δ ppm 16.25 (q, 1C) 23.48 (t, 1C) 24.42 (q, 1C) 26.99 (q, 2C) 77.70 (s,1C) 98.69 (d, 1C) 100.79 (s, 1C) 102.43 (d, 1C) 104.51 (s, 1C) 106.63(d, 1C) 111.67 (s, 1C) 114.35 (d, 1C) 120.63 (s, 1C) 121.30 (d, 1C)126.73 (d, 1C) 131.02 (d, 1C) 131.42 (s, 1C) 152.36 (s, 1C) 156.51 (s,1C) 159.04 (s, 1C) 160.61 (s, 1C) 161.27 (s, 1C) 162.14 (s, 1C) 182.44(s, 1C).

Example 4 Preparation of Organic 70% ETOH Extracts from Morus alba

2 kg of dried Morus alba roots and root barks were cut, crushed, andthen extracted with approximately ten-fold volume (20 L) of 70% ethylalcohol in water (v/v); the extraction was carried on at 80° C. for 5hrs. The ethanol solution was filtered to obtain the supernatant whichwas then concentrated with an evaporator under vacuum at 40° C. Thisextraction and concentration procedure was repeated two times. Theextraction solutions were then combined together and concentrated untilthe volume become 1/25 of the original volume. The concentrated solutionwas dried by vacuum freeze-drying to obtain 283.5 g of Morus alba 70%EtOH extract powder 1-01. The extraction yield was about 14.7% (w/w).

Example 5 Isolation of Mulberroside a from Morus alba ETOH Extracts

A 20 g amount of Morus alba 70% ethyl alcohol extract 1-01 from Example4 was loaded onto silica gel column and the column was eluted with astepwise application of solvent mixture containing linear gradient ofhexane:EtOAc (5:1 to 1:5) to give eight subfractions. Among the eightsubfractions, the 8^(th) fraction was subjected to a RP-HPLC column(YMC-ODS) 5 μm, C18 (250×30 mm) by injection onto a preparative HPLCsystem (JAI, LC-9104, Japan) eluted with 15% Acetonitrile in H₂O in 16.2min with UV wavelength 330 nm to afford Compound 5 (mulberroside A) (191mg).

Compound 5 (mulberroside A, C₂₆H₃₂O₁₄): APCI-MS (m/z) [M+H]⁺ 569.58; UVλ_(max) (MeOH): 217.9, 325.6 nm; ¹H NMR (400 MHz, DMSO-d₆) δ ppm 6.34(brs, 1H) 6.52 (dd, J=8.6, 2.4 Hz, 1H) 6.54 (d, J=2.4 Hz, 1H) 6.57 (s,1H) 6.64 (s, 1H) 6.94 (d, J=16.4 Hz, 1H) 7.22 (d, J=16.4 Hz, 1H) 7.45(d, J=8.6 Hz, 1H); ¹³C NMR (125 MHz, DMSO-d₆) δ ppm 60.58 (t, G-6′)60.62 (d, G-6) 69.56 (d, G-4) 69.63 (d, G-4′ 73.20 (d, G-2′) 73.29 (d,G-2) 76.61 (d, G-3′) 76.61 (d, G-3) 77.00 (d, G-5′) 77.04 (d, G-5)100.39 (s, G-1′) 100.76 (s, G-1) 102.65 (d, C-2′) 103.86 (d, C-3) 105.35(d, C-4′ 106.52 (d, C-5) 107.46 (d, C-6′) 117.86 (s, C-1) 123.47 (d,C-6) 126.00 (d, a) 127.27 (d, b) 139.77 (s, C-1′) 155.86 (s, C-2) 157.96(s, C-4) 158.40 (s, C-5′) 158.92 (s, C-3′)

Example 6 Preparation and HPLC Quantification of Extracts from MorusPlants

Morus samples were collected from different plant parts in differentgeological locations in S. Korea. The dry plant materials were groundinto powder. Morus plant powder (20 grams) was mixed with enoughdiatomaceous earth to fill up a 100 mL extraction cell, and extractedwith 70% Ethanol/water by using ASE 350 Extractor (Extraction condition:Heat=5 minutes, Static=5 minutes, Flush=80 volume, Purge=900 seconds,Cycles=3, Pressure=1500 psi, Temperature=60° C.). After extraction, thesolution was concentrated with an evaporator at 50° C. to produce asolid extract.

The target components Mulberroside A, Oxyresveratrol, Kuwanon G, AlbaninG and Morusin in the Morus extracts were quantified with a Luna C18reversed-phase column (Phenomenex, 10 μm, 250 mm×4.6 mm) in a HitachiHPLC system at 325 nm. The column was eluted with a binary gradient of0.1% Formic acid in water (mobile phase A) and acetonitrile (mobilephase B) at 1 ml/min flow rate and 30° C. column temperature.

TABLE 1 Gradient Table of HPLC Analytical Method Time (min) Mobile phaseA Mobile phase B 0.0 90 10 8.0 85 15 35.0 10 90 35.1 0 100 38.0 0 10038.1 90 10 45.0 90 10

Reference Standard Material 72-1 (Morus 70% EtOH extract 1-01) producedaccording to Example 4 was utilized as the quantification standard. Allextract samples were prepared in a concentration around 5 mg/ml in MeOH.After sonicating for approximately 15 minutes, the sample solution wascooled in a flask to room temperature and filtered through a 0.45 umnylon syringe filter and 20 μl of the sample was injected into thecolumn.

Morus plants were collected from South Korea and China from differentgeological locations in both countries. The HPLC quantification ofMulberroside A, Oxyresveratrol, Kuwanon G, Albanin G and Morusin contentin different species, different plant parts, collected from differentlocations, and at different age of plants, are listed in Tables 2 and 3.The actives have been qualified from Morus root bark, root wood, fineroots, stem bark, branch, branch bark, branch wood, and twigs. There aresmall amounts of stilbene-type compounds—Mulberroside A andOxyresveratrol—detected in Morus leaf.

TABLE 2 Quantification of Active Compounds in Morus Collected from S.Korea. Active Content in Extract (%) Morus Plant Mulberroside Oxy-Kuwanon Albanin Extraction No. Part A resveratrol G G Morusin Yield (%)MK-1 Root bark 10.93 0.07 1.66 0.82 0.55 23% MK-2 Root bark 11.58 0.752.79 1.18 1.21 19% MK-3 Root wood 6.40 2.26 0.58 0.20 0.24 8% MK-4 Fineroot 9.58 2.15 2.98 1.73 1.35 15% MK-5 Stem bark 2.89 0.16 0.27 0.420.48 19% MK-6 Root bark 0.36 0.16 0.23 0.00 0.09 18% MK-7 Root bark13.28 0.00 0.25 0.00 0.00 27% MK-8 Root bark 11.71 0.08 0.63 0.25 0.1521% MK-9 Root bark 17.63 0.48 2.80 0.66 1.56 21% MK10 Root bark 0.280.19 1.70 0.06 0.05 16% MK-11 Leaves 0.54 0.06 0.00 0.00 0.00 23% MK-12Fruit 0.00 0.00 0.00 0.00 0.00 35% MK-13 Branch 3.31 4.07 0.14 0.00 0.189% MK-14 Root bark 12.51 0.39 5.73 2.48 2.42 22% MK-15 Root wood 1.582.52 0.36 0.14 0.12 7% MK-16 Branch 22.46 0.09 0.58 0.00 0.57 15% barkMK-17 Branch 4.95 1.78 0.17 0.00 0.00 5% wood MK-18 Root bark 0.41 0.283.36 0.11 0.18 14%

TABLE 3 Quantification of Active Compounds in Morus Collected from ChinaActive Content in Extract (%) Morus Plant Mulberroside Oxy- KuwanonAlbanin Extraction No. Part A resveratrol G G Morusin Yield (%) MC-1Root bark 1.74 0.10 7.29 6.31 5.38 17% MC-2 Root bark 3.42 0.37 4.691.00 1.97 18% MC-3 Root bark 0.04 0.05 0.34 0.00 0.12 8% MC-4 Root bark0.11 0.60 0.39 0.00 0.14 8% MC-5 Root bark 0.24 0.22 0.73 0.00 0.18 9%MC-6 Root bark 14.07 0.36 2.06 1.29 1.42 20% MC-7 Root bark 9.96 1.012.51 0.73 0.78 12% MC-8 Root bark 0.21 2.64 0.06 0.46 1.40 12% MC-9 Rootbark 5.85 1.44 5.11 2.41 8.70 19% MC-10 Root bark 2.81 0.76 11.43 4.213.82 11% MC-11 Root bark 0.03 0.01 0.40 0.75 0.10 11% MC-12 Fruit 0.000.00 0.00 0.00 0.00 74% MC-13 Leaves 0.00 0.00 0.13 0.00 0.00 20% MC-14Twigs 2.67 0.90 0.06 0.17 0.03 4%

Example 7 HPLC Quantification of Extracts from Morus Root Bark

Ethanol extracts of Morus root barks were obtained from differentgeological locations in China. The contents of four activecomponents—Mulberroside A, Kuwanon G, Albanin G and Morusin—in thoseMorus extracts were quantified with the HPLC method described in Example6. As shown in the Table 4, two Morus extracts (ME-10 and ME-12)contained none of the four active compounds. Three Morus extracts (ME-6,ME-7 and ME-8) contained no Mulberroside A and very small amounts ofprenylated flavonoids (less than 4% as a total of the three compoundspresent). Another four Morus extracts (ME-3, ME-4, ME-5, and ME-14)contained small amounts of prenylated flavonoids (less than 2% as atotal of the three compounds present) and variable amount ofMulberroside A. This Example clearly demonstrates the lack of enrichmentand standardization of stilbene and prenylated flavonoids in regularMorus root bark extracts.

TABLE 4 Quantification of Active Compounds in Morus Extracts from ChinaMorus Active Content in Extract (%) Extract Mulberroside A Kuwanon GAlbanin G Morusin ME-1  20.4 2.17 0.77 1.31 ME-2  22.26 2.57 0.83 1.49ME-3  10.86 0.42 0.17 0.22 ME-4  1.07 0.22 0.13 0.13 ME-5  2.3 0.54 0.270.23 ME-6  0 0.45 0.15 0.95 ME-7  0 0.47 0.16 0.99 ME-8  0 1.32 0.352.08 ME-9  6.7 2.29 0.99 0.91 ME-10 0 0 0 0 ME-11 6.13 2.15 1.02 0.93ME-12 0 0 0 0 ME-13 8 2.8 1.01 1.06 ME-14 6.49 0.85 0.22 0.21

Example 8 Preparation of Morus alba 70% ETOH Extract 10

Dried Morus alba roots and root barks (93.3 kg) were cut, crushed, andthen extracted with approximately seven-fold volume (700 L) of 70% ethylalcohol in water (v/v); the extraction was carried out at 100° C. for 4hrs. The ethanol solution was filtered to obtain the supernatant, whichwas then concentrated with an evaporator under vacuum at 40° C. Thisextraction and concentration procedure was repeated two times. Theextraction solutions were then combined together and concentrated untilthe volume become 1/25 of the original volume. The concentrated solutionwas dried by vacuum freeze-drying to obtain 18.3 kg of Morus alba 70%EtOH extract powder 10. The extraction yield was about 19.6% (w/w). Themajor active component content is listed in Table 5 of Example 12.

Example 9 Preparation of Morus alba ETOAC Fraction 11

Morus alba EtOH extract produced according to Example 8 was extractedwith approximately two-fold volume of ethyl alcohol (EP grade, DucksanChemical, Korea) from 4 kg of dried Morus alba root bark yielded 570 gof Morus alba EtOH extract powder. The EtOH extract was partitioned withhexane and water followed by extraction with ethyl acetate. Extractionwas performed by homogenization of the extraction solution at 15,000 rpmfor five minutes with homogenizer (IKA T25D, Germany). The wellhomogenized extraction solution was then separated by centrifuge(Beckman J-20XP, Germany) at 3,000 rpm (rotor# JLA 8.1000) for fiveminutes. Corresponding n-hexane soluble and water soluble extracts wereprepared from 570 g of the crude Morus alba EtOH powder. This resultedin production of 80.5 g of the n-hexane soluble extract and 156 g of thewater-soluble extract of Morus alba. After solvent partition with EtOAc,the upper layer (EtOAc soluble layer) was filtered by filter paper(Hyundai Micro, No. 20, Korea) and the EtOAc solution was collected. Theresidue (precipitate material) collected from the centrifugation wasre-extracted with two-fold volume (300 L) of ethyl acetate (EP grade,Ducksan Chemical, Korea). The re-extracted solution was agitated at 150rpm for 2 hours. The resulting mixture was then filtered (Hyundai Micro,No. 20, Korea) to obtain an additional EtOAc extract solution. Theabove-described procedure was repeated two times. The three resultingEtOAc extract solutions were combined and concentrated by evaporator at40° C. to obtain the final EtOAc extract 11. The final amount of Morusalba EtOAc fraction 11, obtained from this process was 327 g. The majoractive component content is provided in Table 5 (Example 12).

Example 10 Preparation of Morus alba 70% ETOH Precipitate Extract 12

Morus alba EtOH precipitate extract 12 was produced by follows; 634kilograms (KG) of dried Morus alba roots and root barks were cut,crushed and extracted with approximately 7 fold volume (3600 liters (L))of 70% ethyl alcohol in water (v/v); the extraction solvent was treatedat 80° C., for 4 hrs; the residue was filtered to obtain the supernatantwhich was then concentrated with an evaporator at 40° C. Theabove-described procedure was repeated three times. The extractionsolutions were then concentrated until the volume become about 1/30 theoriginal starting volumes. Then the concentrated solutions were combinedto evaporate again in order to reduce volume of concentrated solutionuntil 1/90 volume of the original extraction solution. The concentratedsolution was rested at room temperature for 24 hours (hr) to allowseparation into two layers (supernatant and precipitate-layer). Theprecipitate was filtered and dried by vacuum freeze-drying to obtain Malba 70% EtOH precipitate powder. A total of 24 kg of the resultingproduct was obtained from 634 kg of raw plant material. The extractionyield was about 3.79% (w/w). The major active component content islisted in Table 5 (Example 12).

Example 11 Preparation of Morus alba 70% ETOH Extract (13-1),Precipitate (13-2), and Supernatant (13-3) Extracts

Morus alba EtOH precipitate extract was produced as follows: 465 kg ofdried Morus alba roots and root bark were cut, crushed, and extractedwith approximately 10-fold volume (4500 L) of 70% ethyl alcohol in water(v/v); the extraction solvent was treated at 80° C. for 4 hrs; theresidue was filtered to obtain the supernatant which was concentratedwith an evaporator at 40° C. Above-described procedure was repeatedthree times. The extraction solutions were concentrated until the volumebecome 1/30 the original volume. The concentrated solutions were thencombined and evaporated again to reduce the volume of the concentratedsolution until 1/90 volume of the original extraction solution wasachieved. The concentrated solution was left at room temperature for 24hr to allow separation into a supernatant and precipitate layer. Theprecipitate layer was then dried by vacuum to obtain 12 kg of Morus alba70% EtOH precipitate powder 13-2. The precipitate yield from Morus rootbarks was about 2.6% (w/w). The supernatant layer was dried by vacuumdrying to obtain 24 kg Morus alba 70% EtOH supernatant powder 13-3. Theextraction yield for the supernatant 13-3 was about 5.2%.

Morus alba 70% EtOH combination extract (13-1) was obtained by blending2 kg of precipitate (13-2) and 4 kg of supernatant (13-3)). The majoractive component content in both Morus alba EtOH extract 13-1,precipitate 13-2 and supernatant 13-3 is listed in Table 5 (Example 12).

Example 12 HPLC Quantification of Active Content in Different Morus albaExtracts

The detailed HPLC quantification method for Mulberroside A,Oxyresveratrol, Kuwanon G, Albanin G and Morusin content was describedin Example 6. Table 5 lists the active contents in different Morus rootbark extracts as prepared in the Examples 8, 9, 10 and 11.

TABLE 5 Quantification of Active Compounds in Morus Extracts PrenylatedFlavonoid in Extract (%) Stilbene in Extract (%) Total MorusMulberroside Oxy- Total Kuwanon Albanin Prenylated Extracts Aresveratrol Stilbenes G G Morusin Flavonoids 10 2.88 1.64 11 1.55 0.331.89 9.31 6.74 6.84 22.89 12 1.27 0 1.27 5.30 4.28 4.25 13.83 13-1 7.310.26 7.57 3.12 1.71 2.01 6.84 13-2 0.76 0 0.76 5.51 3.98 4.48 13.97 13-37.50 0 7.50 1.27 0.36 0.48 2.11

Example 13 Preparation of Organic Extracts from Curcuma longa

A total of 20 grams of dried rhizome powder of Curcuma longa were loadedinto two 100 ml stainless steel tube and extracted twice with an organicsolvent mixture (methylene chloride/methanol in a ratio of 1:1) using anASE 300 automatic extractor at 80° C. and under 1,500 psi of pressure.The extract solution was filtered, collected, and evaporated with arotary evaporator to give crude organic extract (OE) (6.04 g, 30.2%yield).

Example 14 High Throughput Purification (HTP) of Curcuma longa OrganicExtracts

The Curcuma longa organic extract (OE, 400 mg) as described in Example13 was loaded onto a pre-packed flash column (2 cm ID×8.2 cm, 25 ml, 10g silica gel), eluted using a Hitachi high throughput purification (HTP)system with an unique gradient mobile phase of (A) 50:50 EtOAc:hexanesand (B) methanol from 100% A to 100% B in 30 minutes at a flow rate of 5mL/min. A total of 88 fractions were collected in a 96-deep-well plateat 1.9 mL per well using a Gilson fraction collector. The sample platewas dried under low vacuum and centrifugation, and then the driedsamples were resuspended in 1.5 mL dimethyl sulfoxide (DMSO) per well. Aportion (100 μL) from each well was taken and combined (based on UVtrace) for the BKB1 inhibition assay.

Example 15 Bradykinin B1 Radioligand Binding Assay of Curcuma Extractsand Fractions Thereof

Bradykinin B1 (BKB1) radioligand binding assay was conducted todetermine the inhibition activity of Curcuma longa OE and extractfractions on BKB1 binding to BKB1 receptor (BKB1R). Membranes from humanIMR-90 lung fibroblasts, stimulated with IL-1β in modified HEPES buffer(PH=7.4), were incubated with a test sample in the presence of 0.9 nM[³H](Des-Arg¹⁰)-Kallidin for 60 minutes at room temperature. Afterincubation, membranes were filtered and washed five times with modifiedDPBS buffer (pH=7.4). Samples were scintillation counted to determinethe amount of specifically bound to the BKB1 receptor containingmembrane.

The Curcuma longa OE was tested at a concentration of 166 μg/mL and IC₅₀values were determined using the same method with serial dilutions atconcentrations ranging from 400 μg/mL and 5 ng/mL to obtain adose-response curve. Data showing inhibition of BKB1 binding to BKB1R byCurcuma longa OE extracts is provided in Table 6.

TABLE 6 Inhibition of BKB1 Receptor Binding by Curcuma longa OE SampleBKB1 (166 μg/ml) POC (%) BKB1 IC₅₀ (μg/mL) OE extract −0.14 9.6

Curcuma longa OE showed strong inhibition of BKB1 binding with an IC₅₀of about 9.6 μg/mL. Furthermore, HTP fractions of the Curcuma longa OEwere examined in the BKB1 binding assay (data not shown). The activityprofile of the HTP fractions indicates that fractions 11-22, 34, and 38had the most potent BKB1 receptor binding inhibition, with a meanpercentage of control (POC) below 10%. Curcuminoids were found to be themajor active compounds associated with the activity of HTP fractions11-22.

Example 16 BKB1 and BKB2R Binding Activity of Curcuma Compounds

BKB1 binding assay, as described in Example 15, was used to testcurcumin compound isolated from a Curcuma longa extract (Compound 11),as well as commercially available curcumin purchased from Sigma-Aldrich(C1386). Curcumin was tested at final concentrations ranging from 200 μMto 5 nM. Binding curves were plotted by non-linear regression fit (usingGraphPad Prizm software). K_(i) values were computed using Cheng-Prusoffalgorithm. In addition, inhibition of BKB2 receptor binding activity bycurcumin was examined with methods similar to those described in Example15 for the BKB1 receptor with some modifications. Bradykinin RadioligandBinding Assay (BKB2) was conducted using a standard assay under thefollowing conditions:

-   -   1. Composition of Assay Buffer: 24 mM TES, pH 6.8, 1 mM        1.10-Phenanthrioline, 0.3% BSA.    -   2. Source of BKb2R: CHO-K1 cells expressing recombinant human        BKb2R    -   3. Ligand: [³H]-Bradykinin: 0.2 nM.    -   4. Incubation time: 90 min RT.    -   5. Reading: TopCount.

Commercial curcumin (Sigma, C1386) was tested at concentrations rangingfrom 200 μM to 5 nM. Binding curves for commercial curcumin does notconform to mass action law for competitive inhibitor. K_(i) was manuallycalculated by using Cheng-Prusoff equation. The inhibition activity forBKB1 and BKB2 by curcumin is provided in Table 7.

TABLE 7 Inhibition of BKB1 and BKB2 by Curcumin Compound BKB1 Ki (μg/ml)BKB2 Ki (μg/ml) Curcumin 2.173 58

The data indicate that curcumin is a selective BKB1 antagonist since itshows much stronger inhibition of BKB1 binding activity as compared toBKB2 binding.

Example 17 Preparation of Curcuma longa Ethyl Alcohol Extract 19

Curcuma EtOH extract was produced as follows: 20 kg of dried Curcumalonga rhizomes (roots) were pulverized, and extracted with approximately4-fold volume (80 L) of 100% ethyl alcohol and the extraction solventheld at 80-85° C. for 30 hrs. The residue was filtered to obtain asupernatant that was concentrated with an evaporator at 85-90° C. Theextraction solutions were then concentrated until the volume was 1/25 ofthe original volume. The concentrated solution was dried by spray dryprocess (temperature I/P 200° C. and O/P 95° C.) to obtain about 1 kg of25% Curcuma in EtOH extract powder 19 with reddish-orange color. Theextraction yield was about 5% (w/w).

Example 18 Quantification of Curcumin in Curcuma Rhizome Extract

The following analytical method was used to determine the amount ofCurcumin in the Curcuma longa rhizome extracts. An Agilent HPLC/PDAsystem was used with a C18 reversed-phase column (Phenomenex, USA, Luna5 um, 250 mm×4.6 mm) for detection and quantitation of Curcumin andminor components. A binary 0.1% acetic acid in purified water (mobilephase A) and acetonitrile (mobile phase B) gradient was used for elutionof Curcumin components as described in Table 7. The flow rate was set to1 ml/min passing through the Luna C18 column with a column temperatureof 35° C. The UV detector was set to read absorbance at 407 nm.

TABLE 7 Curcumin HPLC Gradient Elution Scheme Time (min) Mobile phase A% Mobile phase B % 0 55 45 10.0 55 45 10.1 10 90 25.0 10 90 25.1 55 4530.0 55 45

The quantification standard—Curcumin was purchased from Sigma-AldrichCo. The highest concentration level of Curcumin was 0.05 mg/ml anddiluted to L5 from L1 (0.0031 mg/ml) using methanol. Concentration ofCurcuma longa rhizome extract samples were adjusted to about 1 mg/ml inmethanol in a volumetric flask and sonicated until dissolved(approximately 20 minutes), then cooled to room temperature, mixed welland filtered through a 0.45 μm nylon syringe filter. Then 10 μl ofsample was quantified by HPLC, which results for Curcuma longa rhizomeextract are provided in Table 8.

TABLE 8 HPLC Quantification of Curcuma longa Rhizome Extract SampleCurcumin % Curcuminoids (total) % 110 16.34 30.04 210 14.71 27.93 31013.08 26.53

Example 19 Preparation of Gambir (Uncaria gambir) Extract 21

Uncaria gambir water extract was produced as follows. 100 kg of driedleaves of Uncaria gambir was cut, crushed, and extracted with 15-foldvolume (1500 L) of 70% ethyl alcohol and the extraction solvent treatedat 80° C. for 7 hrs. The resulting residue was filtered to obtain asupernatant. The above-described procedure was repeated for second time.The extraction supernatant solutions were combined together andconcentrated with an evaporator at 46° C. under vacuum condition untilthe volume became 1/30^(th) of the original volume. The concentratedsolution was evaporated further to reduce volume of concentratedsolution until 1/90 volume of the original solution. The resultingconcentrated solution was then rested at room temperature for 24 hrs toallow precipitate to form in the concentrated solution. The precipitatewas filtered and dried under vacuum to obtain precipitate powder asUncaria gambir extract powder 21. The yield from 100 kg of dried leavesof Uncaria gambir was about 6 kg of extract powder, so the extractionyield was about 6% (w/w).

Example 20 HPLC Quantification of Uncaria gambir Extracts

The following analytical method was used to determine the amount ofcatechin in the Uncaria gambir leaf extracts. An Agilent HPLC/PDA systemwith a C18 reversed-phase column (Phenomenex, USA, Luna 5 um, 250 mm×4.6mm) was used for the detection and quantitation of catechin compound inGambir extracts. A binary column gradient was used for elution ofmaterial from the column. Mobile Phase A: 0.1% phosphoric acid inpurified water, and Mobile Phase B: acetonitrile gradient was used forelution (Table 9). The flow rate was set to 1.0 ml/min passing throughthe Luna C18 column with a column temperature of 35° C. The UV detectorwas set to record absorbance at 275 nm.

TABLE 9 Gradient Table of HPLC Analytical Method Time (min) Mobile PhaseA Mobile Phase B 0.0 85.0 15.0 7.0 85.0 15.0 12.0 10.0 90.0 16.5 10.090.0 16.6 85.0 15.0 24.0 85.0 15.0

Pure catechin reference sample was purchased from Sigma-Aldrich Co. Thereference sample was dissolved in MeOH:0.1% H₃PO₄ (1:1). Highest levelconcentration range of catechin was 0.5 mg/ml and diluted to L5 from L1(0.003 mg/ml) using 50% methanol in 0.1% H₃PO₄. Concentration of theGambir extract samples were adjusted to 2 mg/ml in 50% methanol in 0.1%H₃PO₄ in a volumetric flask and sonicated until dissolved (approximately10 minutes), and then cooled to room temperature, mixed well andfiltered through a 0.45 μm nylon syringe filter. HPLC analysis wasperformed by injecting a 20 μl sample into the HPLC.

TABLE 10 HPLC Quantification of Gambir Extract Sample Catechin % 21020.0 212 18.5

Example 21 Preparation of Acacia catechu 65% Catechin Extract

Acacia catechu 65% catechin extract was produced as follows: 500 kg ofAcacia catechu (KATHA) was put into 750 L of 50% ethyl alcohol andstirred at room temperature for 90 min. After 500 L of ethyl acetate wasput into the homogenized KATHA slurry, it was stirred smoothly for 30min. The slurry was allowed to separate into two layers for 1 hr. Theethyl acetate layer was moved into a new bottle, and the partition wasrepeated with the water layer. Both the 1st and 2nd ethyl acetate layerswere combined and concentrated at 60-62° C. until TDS 30%, and thenspray dried (temp. I/P 190° C.—O/P 90° C.). A total of 72.5 kg Acaciacatechu extract was obtained from 500 kg of raw material with catechinand epicatechin total content at not less than 65%. The extraction yieldwas 14.5% (w/w).

Example 22 Ex Vivo Glycosaminoglycans (GAG) Release Assay

Articular cartilage from hock joints of rabbits (2.5 kg body weight) wasremoved immediately after each animal was sacrificed and articularcartilage explants were obtained by following the method described bySandy et al. (Biochem. Biophy Acta 543:36, 1978). Briefly, after thearticular surfaces were surgically exposed under sterile conditions,approximately 200-220 mg articular surfaces per joint were dissected andsubmerged into complete medium (DMEM, supplemented with heat inactivated5% FBS; penicillin 100 U/ml; streptomycin 100 ug/ml). They were thenrinsed several times with the complete medium and incubated for 1 to 2days at 37° C. in a humidified 5% CO₂/95% air incubator forstabilization. The complete medium was replaced with a basal medium(DMEM, supplemented with heat-inactivated 1% FBS, 10 mM HEPES, andpenicillin 100 U/ml streptomycin 100 μg/ml). Approximately 30 mgcartilage pieces (2×3×0.35 mm/piece) were placed in 24-well plates andtreated with given concentrations of test agents. After pretreatment for1 h, 5 ng/ml of rhIL-1α was added to the culture medium and furtherincubated at 37° C. in a humidified 5% CO₂/95% air incubator. Theculture medium was collected 24 h later and stored at −20° C. untilassay.

The amount of sulphated GAGs (e.g., released from proteoglycans) in themedium at the end of the reaction reflects the amount of articularcartilage degradation, which was determined using the commerciallyavailable 1,9-dimethy-methylene blue method according to theinstructions of the manufacturer (Blyscan™ assay, Accurate Chemical andScientific Corp., Westbury, N.Y.).

Example 23 Effect of Purified Compounds from Morus on Ex Vivo GagRelease

Rabbit cartilage explants were cultured with rhIL-1α (5 ng/ml) in theabsence or presence of purified Morus compounds isolated according toExample 3 to examine the protective effects on proteoglycan (PG)degradation. Purified compound inhibited rhIL-1α-mediated degradation ofPG in a concentration dependent manner. Especially, Mulberroside A,Oxyresveratrol and Morusin showed a strong inhibitory effect whencompared with diclofenac treated group.

TABLE 12 Effect of Morus Compounds on Ex Vivo GAG Release Sample Dose %GAG release Normal — 36.6 IL-1α 5 ng/ml 100 Diclofenac 300 μg/ml 34.6Mulberoside A 25 μg/ml 73.1 50 μg/ml 75.8 100 μg/ml 70.5 Kuwanon G 25μg/ml 56.6 50 μg/ml 48 100 μg/ml 44.4 Oxyresveratrol 25 μg/ml 59.8Morusin 25 μg/ml 48.4 50 μg/ml 49.9 100 μg/ml 33.6

Example 24 Morus Extract Reduces Ex Vivo Gag Release

Rabbit cartilage explants were cultured with rhIL-1α (5 ng/ml) in theabsence or presence of Morus extracts to examine the protective effectson PG degradation. Morus extracts inhibited rhIL-1α-mediated degradationof PG in a concentration dependent manner. All samples showed a strongeffect as compared to that of IL-1α treated group.

TABLE 13 Effect of Morns Extracts on Ex Vivo GAG Release Sample Dose %GAG release Normal / 36.6 IL-1α  5 ng/ml 100 Diclofenac 300 μg/ml 34.613-1 100 μg/ml 50.2 200 μg/ml 41.9 11 100 μg/ml 49.9 200 μg/ml 37.3 13-3100 μg/ml 67.20 200 μg/ml 61.3

Example 25 Preparation of Ethanol Extracts from Mentha piperita

Peppermint (Mentha piperita) 90% EtOH extract (lot# RM604-13002) wasproduced as follows: 73.4 kg of dried Mentha piperita was cut, crushed,and extracted with a 15-fold volume (1100 L) of 90% ethyl alcohol (v/v)at 85° C. for 3 hrs. The resulting residue was filtered to obtain asupernatant that was concentrated with a vacuum evaporator at 40° C. Theresulting residue was extracted a second time with 13-fold volume (950L) of 90% ethyl alcohol (v/v) at 40° C. for 1 hrs and filtered to obtaina second supernatant which was concentrated with a vacuum evaporator at40° C. The resulting concentrated cake was dried under vacuum to obtain19.3 kg of Peppermint 90% EtOH extract powder designated as Extract 25.The extraction yield was 25.3% (w/w).

Example 26 Preparation of Methanol and Other Organic Extracts fromMentha piperita

Dried ground peppermint leaf powder (Mentha piperita) (21.7 g) loadedinto two 100 ml stainless steel tubes and extracted twice with anorganic solvent mixture (methanol) using an ASE 300 automatic extractorat 80° C. under a pressure of 1,500 psi. The extract solution wasautomatically filtered, collected, and evaporated with a rotaryevaporator to give a crude organic extract (ME 26-1) (4.48 g, 20.64%yield).

Alternatively, 252.3 g of dried ground leaf powder of Mentha piperitawas extracted with methanol three times by refluxing one hour each time.The organic solution was combined and evaporated under vacuum to providemethanol extract (ME 26-2) 40.88 g with a yield of 16.20%.

Similar results were obtained using the same procedure, but with theorganic solvent being replaced with methanol or ethanol to provide amethanol extract (ME) or ethanol extract (EE), Ethanol:H₂O (7:3)extracts, Ethanol:H₂O (1:1) extracts, Ethanol:H₂O (3:7) extracts andwater extracts respectively.

Example 27 Effect of Curcuma and Uncaria Extracts on Ex Vivo Gag Release

Rabbit cartilage explants were cultured with rhIL-1α (5 ng/ml) in theabsence or presence of Curcuma extract from Example 17 or Uncariaextract from Example 19 to examine the protective effect on PGdegradation. Curcuma extract 19 decreased rhIL-1α-mediated degradationof PG in a concentration dependent manner, while Uncaria extract 21showed a weak protective effect on PG degradation.

TABLE 14 Effect of Curcuma and Gambir Extracts on Ex Vivo GAG ReleaseSample Dose % GAG release (-) — 39.0 IL-1α   5 ng/ml 100.0 Diclofenac 300 μg/ml 45.6 19   30 μg/ml 88.9 (Curcuma)   50 μg/ml 65.0 66.7 μg/ml59.2  100 μg/ml 38.2  300 μg/ml 50.4 21 66.7 μg/ml 97.7 (Gambir)   80μg/ml 81.0  100 μg/ml 78.0  120 μg/ml 86.4  200 μg/ml 88.4  300 μg/ml88.4

Example 28 Effect of Peppermint Extract on Ex Vivo Gag Release

Rabbit cartilage explants were cultured with rhIL-1α (5 ng/ml) in theabsence or presence of Peppermint extract from Examples 25 and 26 toexamine the protective effects on PG degradation.

TABLE 15 Effect of Peppermint Extracts on Ex Vivo GAG Release SampleDose % GAG release Normal — 34.5 IL-1α  5 ng/ml 100 Diclofenac 300 μg/ml22.6 191-8 150 μg/ml 110.9 250 μg/ml 84.1 500 μg/ml 73.0 622-9 150 μg/ml91.5 250 μg/ml 79.2 500 μg/ml 68.7

Peppermint extract inhibited rhIL-1α-mediated degradation of PG in aconcentration dependent manner, although the effect of Peppermintextracts on PG degradation were weaker than the diclofenac treatedgroup.

Example 29 Effect of Curcuma longa (C):Morus (M) Compositions on Ex VivoGag Release

Rabbit cartilage explants were cultured for 24 hr with rhIL-1α (5 ng/ml)in the absence or presence of a mixture of Curcuma and Morus extracts toexamine the protective effects on PG degradation. The plant extractsfrom Morus and Curcuma were produced according Examples 8 and 17,respectively. Curcuma and Morus extracts were combined at differentratios, including 4:1, 2:1, 1:1, 1:2 and 1:4, respectively. Thecompositions were tested at four doses—50, 100, 200 and 300 μg/ml. Asshown in Table 17, all compositions of plant extracts prevented rhIL-1αmediated degradation of articular cartilage in a concentration dependentmanner.

TABLE 17 Effect of Morus/Curcuma Compositions on Ex Vivo GAG ReleaseSample Dose (μg/ml) % GAG release (-) — 51.9 IL-1α 0.005 100.0Diclofenac 300 36.8 4C:1M 50 80.5 100 58.1 200 49.1 300 61.8 2C:1M 5082.0 100 57.5 200 47.4 300 68.4 1C:1M 50 88.7 100 62.0 200 54.2 300 59.71C:2M 50 81.6 100 59.5 200 58.0 300 57.2 1C:4M 50 62.6 100 63.3 200 56.7300 32.7

Example 30 Evaluation of Curcuma (C):Morus (M) Composition Synergy on ExVivo GAG Release

Rabbit cartilage explants were cultured for 24 hr with rhIL-1α (5 ng/ml)in the absence or presence of compositions of Curcuma extract, Morusextract, or a mixture thereof to examine the presence of a protectiveeffect on PG degradation. The plant extracts from Morus and Curcuma wereproduced according Examples 8 and 17, respectively. Curcuma and Morusextracts were combined at different ratios, including 1:2 and 1:4. Thecompositions were tested at two doses—200 and 300 μg/ml, or at onedose—75 μg/ml to examine whether the combined extracts workedsynergistically or additively. The individual extract compositions weretested at concentrations that were in proportion to the weight contentof those extracts in the mixed composition.

TABLE 18 Synergistic Effect of C:M Composition versus C or M Alone % %Cmpsn μg/ml Inhibition Cmpsn μg/ml Inhibition Remark 1C:4M 200 85.11C:4M 300 97.8 Theoretical value 1C:4M 200 87.8 1C:4M 300 100Experimental result C 40 49.1 C 60 72.6 Individual M 160 70.7 M 240 92Individual 1C:2M 200 81.7 1C:2M 300 95.6 Theoretical value 1C:2M 20095.8 1C:2M 300 100 Experimental result C 66.7 59.9 C 100 85 Individual M133.3 54.3 M 200 70.6 Individual 1C:1M 75 53 Theoretical value 1C:1M 7557.5 Experimental result C 37.5 33 Individual M 37.5 29.9 Individual

Compositions of Curcuma and Morus extracts interfered with therhIL-1α-mediated degradation of PG in a concentration dependent andsynergistic manner. Especially, compositions 1C:4M (5 wt % curcuminoids,2.4 wt % prenylated flavonoids, 2.4 wt % stilbenes) and 1C:2M (8.3 wt %curcuminoids, 2 wt % prenylated flavonoids, 2 wt % stilbenes) showed asynergistic effect at 200 and 300 μg/ml. Composition 1C:1M (12.5 wt %curcuminoids, 1.5 wt % prenylated flavonoids, 1.5 wt % stilbenes) alsoshowed a synergistic effect at 75 μg/ml. Synergy values were calculatedby using the COLBY formular (Colby, Weeds 15:20, 1967).

Example 31 Effect of Curcuma (C):Morus (M):N-Acetyl Glucosamin (NAG)Compositions on Ex Vivo GAG release

Rabbit cartilage explants were cultured for 24 hr with rhIL-1α (5 ng/ml)in absence or presence of composition of Curcuma and Morus extract toexamine the protective effects on PG degradation. The plant extractsfrom Morus and Curcuma were produced according to Examples 8 and 17,respectively. Curcuma and Morus extracts were combined with N-AcetylGlucosamine (NAG) at a ratio 1C:1M:2NAG. The compositions were tested atfour doses-50, 100, 200 and 300 μg/ml. The individual extracts in thecompositions were tested at concentrations that were in proportions ofthe weight contents of those extracts in the compositions. Synergyvalues were calculated by using the Colby formular (Colby, Weeds 15:20,1967).

TABLE 19 Effect of Curcuma, Morus, and NAG Compositions Sample Dose %GAG release Normal — 40.7 IL-1α   5 ng/ml 100.0 Diclofenac  300 μg/ml30.1 1C:1M:2NAG   50 μg/ml 83.2  100 μg/ml 59.7  200 μg/ml 52.7  300μg/ml 46.4 Curcuma 12.5 μg/ml 71.8   25 μg/ml 74.9   50 μg/ml 50.8   75μg/ml 58.4 Morus 12.5 μg/ml 76.3   25 μg/ml 77.7   50 μg/ml 70.9   75μg/ml 70.9 NAG   25 μg/ml 95.7   50 μg/ml 99.2  100 μg/ml 87.5  150μg/ml 81.2

As shown in the Table 19, the composition of plant extracts preventedwith the rhIL-1α mediated degradation of articular cartilage in aconcentration dependent manner. In particular, a 1C:1M:2NAG compositionshowed an unexpected synergistic effect at 300 μg/ml as compared to thethree individual extracts alone (Table 20).

TABLE 20 Synergistic Effect of C:M:NAG Compositions Sample Dose %Inhibition Remark 1C:1M:2NAG 300 μg/ml 89.6 Theoretical value 1C:1M:2NAG300 μg/ml 90.5 Experimental result C  75 μg/ml 70.1 Individual M  75μg/ml 49.2 Individual NAG 150 μg/ml 31.8 Individual

Example 32 Formulation of Morus Root Bark Extract with Other Actives andIngredients

The bone density compositions were formulated with Morus root barkextracts with other active and/or excipient ingredients set forth in thefollowing compositions.

Supplement Facts Serving Size: Two (2) Tablets per day

Directions: Take one (1) Tablet twice daily with meals.

Composition 1

Amount per Serving Amount % Daily Value Glucosamine Hydrochloride 1,500mg * Chinese mulberry extract   200 mg * (Morus alba) (root bark)Curcumin extract   200 mg * (Curcuma longa) (rhizome) Vitamin D3 (ascholecalciferol)   400 IU 100% Calcium (elemental)   600 mg  60% * DailyValue not established

Composition 2

Amount per Serving Amount % Daily Value Glucosamine Sulfate 1,500 mg *Chondroitin sulfate 1,200 mg * Chinese mulberry extract   100 mg *(Morus alba) (root bark) Acacia extract (Acacia catechu)   50 mg *(heartwood and barks) Vitamin D3 (as cholecalciferol)   400 IU 100%Calcium (elemental)   300 mg  30% * Daily Value not established

Composition 3

Amount per Serving Amount % Daily Value N-Acetyl-Glucosamine 1,000 mg *Chondroitin sulfate   200 mg * Chinese mulberry extract   150 mg *(Morus alba) (root bark) Gambir extract (Uncaria gambir)   150 mg *(whole plant) Vitamin D   800 IU  200% Calcium Citrate (elemental) 1,000mg  100% Magnesium Citrate   300 mg   75% Vitamin K1   100 mcg  125%Vitamin K2   10 mcg 12.5% * Daily Value not established

Composition 4

Amount per Serving Amount % Daily Value Chinese mulberry extract   250mg * (Morus alba) (root bark) Curcumin (Curcuma longa)   250 mg *(rhizome) Vitamin D3   800 IU 200% Calcium (elemental) 1,000 mg 100%Magnesium Citrate   400 mg 100% Phosphorus   300 mg  30% Boron (Citrate)  200 mcg * Zink (Citrate)   13 mg  87% * Daily Value not established

Other ingredients: microcrystalline cellulose, croscarmellose sodium,hydroxpropyl cellulose, stearic acid, coating silicon dioxide, glycerin,hydroxypropyl methylcellulose.

This composition provides support of bone and bone and cartilage health.It prevents bone loss, increase bone density and provides bone andcartilage comfort by rejuvinating cartilage, lubricating connectivetissue and strengthening bones. The composition helps you to keep movingand to stay strong and flexible. Bone & Joint Support provides 4-wayprotection for your joints and bones:

-   -   Glucosamine Hydrochloride is provided to protect and strengthen        joints*.    -   Morus alba bioflavonoids are provided to act as potent        antioxidants, to protect joints against harmful inflammation and        oxidants that breakdown bone and cartilage tissues*.    -   Vitamin D3 is provided for enhanced mineral absorption to help        support and nourish bones*.    -   Calcium is provided to improve bone strength and increase bone        density*.

Example 33 Mono-Iodoacetate (MIA) Induced Osteoarthritis Model

Osteoarthritis (OA) is a degenerative joint disease characterized byjoint pain and a progressive loss of articular cartilage and, to date,with no cure. As the disease advances, the biochemical alterations thatoccur within the articular cartilage will result in imbalances betweenanabolic and catabolic processes that ultimately alter the overall jointstructure and function, and lead to chronic pain. Multiple animal modelshave been developed and utilized to study the pathogenesis of OA and toevaluate the effectiveness of novel therapeutic agents with limitedsuccess. An animal model with a robust induction and reproducibility ofjoint pathology, along with pain associated with the disease, wasdesired, so the minimally invasive mono-iodoacetate (MIA) induced OAmodel was employed. Mono-iodoacetate (MIA) is an inhibitor ofglyceraldehyde-3-phosphate dehydrogenase activity shown to inducechondrocyte death and hence reproduces cartilage lesions with loss ofproteoglycan matrix and functional joint impairment similar to human OA(Marker and Pomonis, Methods Mol. Biol. 851:239, 2012).

Male Sprague-Dawley (SD) rats weighing about 170 to about 230 g (6 weeksof age) were purchased and acclimated for one week. One day beforedisease induction, animals were randomized into four group as follows:G1 (Normal), G2 (Vehicle), G3 (Diclofenac; 10 mg/kg) and G4 (G:M; 500mg/kg). Each group was orally gavaged with their respective treatment.Anesthetized rats were injected with 0.8 mg of MIA in a 50 μl salinesolution into the intra-articular pocket one hour after the second doseof treatments. Pain sensitivity was measured once a week using aRandall-Salitto meter and treatment lasted for 6 weeks. Body weightswere measured once a week to calculate the respective weekly dosage ofeach group. Once the in-life study was concluded, structural andcellular alterations of joint tissues as a result of disease progressionand/or treatment efficacy was assessed by using histopathology with amodified Mankin scoring system.

Example 34 Bone Histomorphometry in Therapeutic MIA InducedOsteoarthritis Model

Male Sprague-Dawley (SD) rats weighing 170-230 g (6 weeks of age) werepurchased and acclimated for one week. One day before disease induction,animals were randomized into four group of Normal, Vehicle, Diclofenac(10 mg/kg) and GM (500 mg/kg). Anesthetized rats were injected with 0.8mg of MIA in 50 μl saline solution into the intra-articular pocket. Oneweek after MIA induction, samples were administrated daily with gastrictube for 6 weeks.

Bone histomorphometry was evaluated on both femur and tibia per kneejoint in rats from the MIA-induced osteoarthritis model by Micro CT scanusing an Inveon™ unit (Siemens Healthcare USA, Inc., Pennsylvania, USA)at Korea Basic science institute, Ochang, Korea. BMD was used as anindicator of osteoporosis and fracture risk; BV/TV as an indicator oftrabecular bone volume refers to the ratio of the trabecular bone volumeto the total volume; BS/BV as an indicator of trabecular turnover refersto the ratio of the trabecular bone surface to the trabecular bonevolume of a region of interest.

As shown Table 21, BMD in the GM group was significantly increased whencompared with that of vehicle group. BV/TV showed the increasingtendency in G:M group when compared to that of vehicle group. Diclofenac(positive control) group showed decreasing tendency in BV/TV (Table 21).

TABLE 21 Change in BMD and Bone Architecture in Therapeutic MIA ModelDiclofenac GM Normal Vehicle (10 mg/kg) (500 mg/kg) BMD (mg/cm3) 1023.5± 8.3  881.9 ± 64.0 896.2 ± 29.4 956.2 ± 43.7* BV/TV (%)  74.76 ± 2.6068.24 ± 5.03 65.35 ± 7.75 70.12 ± 4.97  BS/BV  7.22 ± 0.65  8.76 ± 1.13 9.11 ± 1.41 8.22 ± 1.09 Data represented as mean ± SD. * p <0.05 (vsvehicle). BV/TV; total trabecular bone volume /total tissue (bone +marrow) volume

Example 35 Bone Histomorphometry in Concurrent MIA-InducedOsteoarthritis Model

Male Sprague-Dawley (SD) rats weighing 170-230 g (6 weeks of age) werepurchased and acclimated for one week. One day before disease induction,animals were randomized into four group of Normal, Vehicle, Diclofenac(10 mg/kg) and G:M (500 mg/kg). Anesthetized rats were injected with 0.8mg of MIA in 50 μl saline solution into the intra-articular pocket at 1hour after sample treatments. And then samples were administrated dailywith gastric tube for 6 weeks.

Bone histomorphometry was evaluated on both femur and tibia per kneejoint by Micro CT scan using an Inveon™ unit (Siemens Healthcare USA,Inc., Pennsylvania, USA) at Korea Basic science institute, Ochang,Korea. BV/TV as an indicator of trabecular bone volume is the ratio ofthe trabecular bone volume to the total volume. BMD used as an indicatorof osteoporosis and fracture risk. Therefore, we checked those markerswhether GM has the efficacy on the osteoporosis.

As shown Table 22, BMD in both Diclofenac and G:M group wassignificantly increased when compared to that of vehicle group. BothDiclofenac and GM groups had tendency to increase BV/TV values incomparison against vehicle group in MIA rats. (Table 22).

TABLE 22 Change in BMD and Bone Architecture in Concurrent MIA-InducedModel Diclofenac GM Normal Vehicle (10 mg/kg) (500 mg/kg) BMD (mg/cm3)1023.5 ± 8.3  881.9 ± 64.0 982.3 ± 19.4^(#) 996.1 ± 11.6^(#) BV/TV (%) 74.76 ± 2.60 68.24 ± 5.03 71.89 ± 5.43  70.84 ± 5.92  Data representedas mean ± SD. * p <0.05 (vs vehicle). BV/TV; total trabecular bonevolume/total tissue (bone + marrow) volume

Example 36 Ovariectomized (OVX) Osteoporosis Model

CM (a proprietary blend of Curcuma longa and Morus alba extracts) wastested to show the improvement of bone mineral density (BMD) inovariectomized (OVX) rats. Briefly, female Sprague-Dawley (SD) ratsweighing 190-220 g (7 weeks of age) were purchased and acclimated forone week. Rats were randomly divided into ovariectomy (OVX) and shamoperation group (n=10). OVX was performed through flank incision underisoflurane anesthesia. Similar surgical procedures were applied to thesham group except the ovaries were not removed.

OVX rats were divided into OVX control group, Moms-, Curcumin- andC:M-treated groups at three dosages. Ten weeks after the OVX or shamoperation, all samples were administrated daily for 12 weeks. Sham andOVX control rats received the same volume of 0.5% carboxymethylcellulose (CMC) as vehicle. Osteoporosis related parameters includingBMD, total trabecular bone volume/Total tissue (BV/TV), trabecularthickness (TH/TB) were analyzed.

TABLE 23 Change of BMD and architecture in therapeutic MIA inducedmodel. Diclofenac G:M Normal Vehicle (10 mg/kg) (500 mg/kg) BMD (mg/cm3)1023.5 ± 8.3  881.9 ± 64.0 896.2 ± 29.4 956.2 ± 43.7* BV/TV (%)  74.76 ±2.60 68.24 ± 5.03 65.35 ± 7.75 70.12 ± 4.97  BS/BV  7.22 ± 0.65  8.76 ±1.13  9.11 ± 1.41 8.22 ± 1.09 Data represented as mean ± SD. * p<0.05(vs vehicle). BV/TV; total trabecular bone volume/total tissue(bone + marrow) volume

Example 37 Bone Histomorphometry in Concurrent MIA-InducedOsteoarthritis Model

Male Sprague-Dawley (SD) rats weighing 170-230 g (6 weeks of age) werepurchased and acclimated for one week. One day before disease induction,animals were randomized into four group of Normal, Vehicle, Diclofenac(10 mg/kg) and GM (500 mg/kg). Anesthetized rats were injected with 0.8mg of MIA in 50 μl saline solution into the intra-articular pocket at 1hour after sample treatments. Samples were administrated daily withgastric tube for 6 weeks.

Bone histomorphometry was evaluated on both femur and tibia per kneejoint by Micro CT scan using an Inveon™ unit (Siemens Healthcare USA,Inc., Pennsylvania, USA) at Korea Basic science institute, Ochang,Korea. BV/TV as an indicator of trabecular bone volume is the ratio ofthe trabecular bone volume to the total volume. BMD used as an indicatorof osteoporosis and fracture risk.

As shown Table 24 and FIG. 1, BMD in both Diclofenac and GM group wassignificantly increased when compared to that of vehicle group. BothDiclofenac and GM groups had tendency to increase BV/TV values incomparison against vehicle group in MIA rats (Table 24; FIG. 1).

TABLE 24 Change in BMD and Bone Architecture in Concurrent MIA-InducedModel Diclofenac GM Normal Vehicle (10 mg/kg) (500 mg/kg) BMD 1023.5 ±8.3  881.9 ± 64.0 982.3 ± 19.4^(#) 996.1 ± 11.6^(#) (mg/cm3) BV/TV 74.76 ± 2.60 68.24 ± 5.03 71.89 ± 5.43  70.84 ± 5.92  (%) Datarepresented as mean ± SD. * p <0.05 (vs vehicle). BV/TV; totaltrabecular bone volume/total tissue (bone + marrow) volume

Example 38 Clinical Trial Evaluating the Effect of Morus alba and Acaciacatechu on Adults with Osteoarthritis

This study examined the effect of an Acacia/Morus (1A:2M) composition ondiscomfort (onset and overall) and overall function when taken for a12-week period by individuals having osteoarthritis (OA) of the knee. Inthis study, 135 adults, aged 35 to 75 years, with BMI of less than 35kg/m² who had knee pain for at least 15 of the 30 days prior to startingthe study, had symptoms of knee pain for at least 6 months prior tostarting the study, and had a Kellgren-Lawrence grade of I, II, or IIIaccording to a screening X-ray and met all the inclusion/exclusioncriteria were enrolled after signing the informed consent. The studylasted approximately 12 weeks, with subjects seen at a screening visitand 6 study visits (at days 0, 7, 14, 28, 56 and 84). The screeningvisit also included an X-ray of the knee for determination of OA withKellgren-Lawrence grades of I-III inclusionary (unless subject had anX-ray from the past 6 months). At the screening visit, subjects wereprovided with rescue medication (acetaminophen) and were asked to bringthe unused rescue medication to each follow-up visit so that rescuemedication usage could be determined.

The study subjects were randomized and administered one of the followingthree study articles:

Test Product—was comprised of Morus alba (White Mulberry) and Acaciacatechu (Senegalia catechu) (100 mg per capsule), formulated withpharmaceutically acceptable carriers or excipients, includingmicrocrystalline cellulose, magnesium stearate (vegetable) and silicondioxide.

Positive Control—was comprised of a combination of glucosamine (375 mg)and chondroitin (300 mg), formulated with pharmaceutically acceptablecarriers or excipients, including magnesium stearate (vegetable) andsilicon dioxide.

Placebo—was comprised of pharmaceutically acceptable carriers orexcipients, including microcrystalline cellulose, magnesium stearate(vegetable) and silicon dioxide

Subjects were instructed to take two capsules with a morning meal andtwo capsules with an evening meal with up to 8 ounces of water, for atotal of four capsules per day.

One efficacy measure was to determine the effect on discomfort when TestProduct was used for 12 weeks as compared to Positive Control(glucosamine-chondroitin) and Placebo, as measured by: (a) WesternOntario and McMaster University Osteoarthritis Index (WOMAC) painsub-score; (b) Visual Analog Scale (VAS)-Discomfort ratings (ratingsover 12 weeks); and (c) rescue medication use (over 12 weeks).

A second efficacy measure was to determine the acute effect ondiscomfort over the first 7 days of use of Test Product as compared toPositive Control and Placebo, as measured by: (a) Visual Analog Scale(VAS)-Discomfort ratings (daily ratings over the first 7 days of productuse); and (b) rescue medication use (over the first 7 days of productuse).

A third efficacy measure was to determine the effect on overall functionwhen Test Product was used for 12 weeks as compared to Positive Controland Placebo, as measured by: (a) Western Ontario and McMaster UniversityOsteoarthritis Index (WOMAC) stiffness and activities of daily livingsub-scores; (b) Range of Motion Goniometer Testing; and (c) Six MinuteWalk Test (6MWT).

A fourth efficacy measure was to determine the effects on inflammationand bone metabolism when Test Product was used for 12 weeks as comparedto Positive Control and Placebo, as measured by production of thefollowing various biomarkers: (a) tumor necrosis factor-alpha (TNFα);(b) Interleukin-1 beta (IL-1β), (c) Interleukin-10 (IL-10), and (d)urinary CTX-II.

The main efficacy analysis was conducted on a Per-Protocol (PP) basis,using the PP population because the attrition was well within theexpected limit, so no intention-to-treat (ITT) analysis was necessary.For each continuous efficacy variable, the mean change from baseline toeach subsequent time point within each product group was tested fornominal significance by the paired Student t test, or by thenon-parametric Wilcoxon test if substantially non-normally distributed.For each continuous efficacy variable at each time point, and forchanges from baseline to each subsequent time point, the values weretested for an overall difference between the three products by theone-way analysis of variance (ANOVA). For the analysis of the biomarkers(TNFα and CTX-II/CR), three approaches were used as follows: (1)non-parametric testing for any difference between the three groups(using the non-parametric Kruskal-Wallis test instead of the 1-wayANOVA); (2) parametric (ANOVA) and non-parametric (KW) testing betweenthe three groups on the logarithms of the values; and (3) analysis ofcovariance (ANCOVA), for comparing mean changes from baseline betweenUP1306 and G+C, and between UP1306 and Placebo, adjusted for baselinevalue) on the logarithms of the values.

Safety of daily supplementation with UP1306 was determined based onchanges from baseline to 12 weeks in blood work (comprehensive metabolicpanel, complete blood count with differential and PT/INR), bloodpressure (BP), heart rate (HR), adverse events, and subjective remarks.

All safety analyses were conducted on the Safety population. For eachcontinuous safety variable, the mean change (or mean percentage change)from baseline to each subsequent time point within each product groupwas tested for significance by the paired Student t test, or by thenon-parametric Wilcoxon test if substantially non-normally distributed.For each continuous safety variable at each time point, the meandifferences in the variable, and in the change in that variable frombaseline, between the three different products was tested forsignificance by the one-way analysis of variance (ANOVA). For eachcategorical safety variable, the difference in the distribution ofcategories between the different test articles was tested forsignificance by the Fisher Exact test if possible, or by the Chi-Squaretest if necessary. Adverse events (AEs) were listed, MedDRA encoded,grouped by general type of event (gastrointestinal, neurologic, cardiac,etc.), and cross-tabulated by event type and product group. Differencesin AE patterns between test articles were measured by the Fisher Exacttest. Subjective remarks were categorized to the extent possible, andanalyzed for pattern differences between product groups in the same wayas AEs.

Results Safety

There were no changes of clinical significance for any of the safetyendpoints (e.g. blood pressure, heart rate and safety lab values). Therewas also no significant association between the test product andfrequency of occurrence of adverse events. No serious adverse events(SAEs) were observed during the course of this study.

A total of 43 adverse events (AEs) were observed among 30 of the 133subjects in the Safety population. Fifteen (15) of the 43 AEs wereobserved in the subjects in the Test Product (Morus/Acacia) group, ten(10) of the 43 AEs were observed in the subjects in the Positive Control(glucosamine and chondroitin combination) group, and 18 of the 43 AEswere observed in the subjects in the Placebo group.

Fourteen (14) of the 43 total AEs, among seven subjects, were consideredprobably or possibly related to the Test Product; the other one wasconsidered unlikely or definitely not related to the Test Product. Amongthe 14 probably or possibly related AEs, three (3) AEs occurred amongtwo of the subjects in the Test Product group; two AEs occurred amongtwo of the subjects in the Positive Control group; and 9 AEs occurredamong three of the subjects in the Placebo group.

Overall, there were no safety concerns raised with the Test Product.

Efficacy

There were significant improvements from baseline to most time-pointsfor all efficacy measures within groups. The two significant differencesobserved between the Test Product and the controls were the following:

-   -   WOMAC Pain: significant decrease from baseline to Day 56 for        Test Product over the Positive Control (p=0.048).    -   Urine CTX-II: significant difference between the changes for        Test Product and Placebo after 12 weeks of use (p=0.029).

Based on the data from this study, the high rate of response for thePlacebo group made it difficult to extract statistically significantdata between the three groups as the response rate was favorable for allthree study groups. The subject reported outcomes (WOMAC, VAS) failed toshow statistical significance other than one time point for Test Product(Day 56 for UP1306 over the Positive Control (p=0.048)), but theobjective biomarkers, such as CTXII, met the objective by showingsignificant difference between the Test Product and Placebo after 12weeks product use (p=0.029).

As noted above, the measure of the following biomarkers: (a) tumornecrosis factor-alpha (TNFα); (b) Interleukin-1 beta (IL-1β), (c)Interleukin-10 (IL-10), and (d) urinary CTX-II, was used as a surrogatemeasure of the effect Test Product had on inflammation and bonemetabolism when used for 12 weeks, as compared to Positive Control andPlacebo. The values for interleukin-1 beta (IL-1β), interleukin-10(IL-10) were below the limits of quantitation for the majority of thesubjects resulting in a small data set; therefore, these were notanalyzed. In addition, tumor necrosis factor-alpha (TNFα) showed nostatistically significant changes in scores within group for any of thethree tested articles, including no statistically significant differencein the change from baseline to day 84 between groups (data not shown).

Measurement of Urinary CTX-II at Baseline and at Day 84

CTX-II measurements Urinary levels of collagen type II C telopeptidefragments were measured by the CartiLaps ELISA assay. The assay uses ahighly specific monoclonal antibody for the detection of degradationproducts of C-terminal telopeptide of type II Collagen. The assay isbased on the competitive binding of the monoclonal antibody to urinaryfragments of type II collagen.

The concentration of the CTX-II ELISA (ng/l) was standardized to thetotal urine creatinine (mmol/l): concentration/creatinine=ng/mmol.Creatinine concentration was measured using a creatinine Colorimetricassay kit (Cayman Chemical) that relies on the Jaffe's reaction. CTX-IIbiomarker values were not normally distributed and consequently the meanvalues and standard deviations were calculated by non-parametricstatistics.

TABLE 1 Urinary CTX-II/CR (ng/mmol), by Product, by Visit, all Subjects(Actual Values)* Visit Test Product Positive Control Placebo Visit 2 414± 231 (44) 403 ± 310 (45) 339 ± 208 (44) Randomization 364 (131-1,117)342 (70-1,894) 263 (92-888) Visit 7 Day 84 377 ± 296 (44) 404 ± 233 (45)417 ± 266 (43) 301 (87-1,890) 357 (48-1,216) 305 (126-1,129) Change from−37 ± 319 (44)  2 ± 303 (45)  85 ± 238 (43) Baseline to −23 (−667-1,372) 9 (−1,174-605)  6 (−277-791) Day 84 *analysis using log transformedvalues

TABLE 2 Urinary CTX-II/CR (ng/mmol), by Product by Visit, in PPPopulation 3-group Comp ANCOVA Non- TP par par vs. TP vs. Visit TestProduct Positive Control Placebo ANOVA KW PC Pla Visit 2  5.88 ± 0.53(43) 5.77 ± 0.67 (44) 5.68 ± 0.60 (41) 0.332 0.340 Rand  5.89(4.88-7.02) 5.82 (4.25-7.55) 5.61 (4.53-6.79) (np) Visit 7  5.74 ± 0.61(43) 5.81 ± 0.64 (44) 5.91 ± 0.57 (40) 0.428 0.499 Day 84  5.71(4.47-7.54) 5.86 (3.87-7.1) 5.75 (4.9-7.03) (np) Change −0.14 ± 0.62(43) 0.04 ± 0.66 (44) 0.25 ± 0.64 (40) 0.025 0.070 0.327 0.029 from−0.08 (−1.69-1.3) 0.03 (−1.26-1.6) 0.13 (−0.75-2.03) (np) Rand to p =0.165 (np) p = 0.804 (np) p = 0.042 (np) Day 84 *analysis using logtransformed values

There was a statistically significant difference between the changes forTest Product and Placebo after 12 weeks of product use (p=0.029).

Urinary CTX-II levels are useful for detecting populations at high riskof joint damage progression early in the disease. These data alsoindicate that in these patients with increased bone/cartilagedegradation, even in the absence of severe joint damage, earlyintervention with products aimed at reducing both bone and cartilagedegradation—in combination, for example, with an anti-inflammatorytherapy—may help to prevent subsequent joint damage. These findings haveimportant clinical implications for the management of OA, RA and othercartilage degradation related conditions.

The various embodiments described above can be combined to providefurther embodiments. All of the U.S. patents, U.S. patent applicationpublications, U.S. patent applications, foreign patents, foreign patentapplications and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheet areincorporated herein by reference, in their entirety. Aspects of theembodiments can be modified, if necessary to employ concepts of thevarious patents, applications and publications to provide yet furtherembodiments.

These and other changes can be made to the embodiments in light of theabove-detailed description. In general, in the following claims, theterms used should not be construed to limit the claims to the specificembodiments disclosed in the specification and the claims, but should beconstrued to include all possible embodiments along with the full scopeof equivalents to which such claims are entitled. Accordingly, theclaims are not limited by the disclosure.

1. A composition for bone health, cartilage health or both, comprising amixture of a Morus extract enriched for one or more prenylatedflavonoids and one or more Stilbenes, and an Acacia extract enriched forflavans.
 2. The composition according to claim 1, wherein the Morusextract and the Acacia extract are blended in a 2:1 weight ratio.
 3. Thecomposition according to claim 1, wherein the Morus extract is fromMorus alba, and the Acacia extract is from Acacia catechu.
 4. Thecomposition according to claim 1 wherein the Acacia extract is comprisedof 0.01% to 99.9% of flavans.
 5. The composition according to claim 1wherein the Morus extract is comprised of 0.1% to 49.9% of prenylatedflavonoids.
 6. The composition according to claim 1 wherein the Morusextract is comprised of 0.1% to 49.9% of Stilbenes.
 7. The compositionaccording to claim 1, wherein the one or more prenylated flavonoids isAlbanin G, Kuwanon G, Morusin, or any combination thereof.
 8. Thecomposition according to claim 1, wherein the one or more Stilbenes isoxyresveratrol, mulberroside A, or a combination thereof.
 9. Thecomposition according to claim 1, wherein the major active ingredientsin the Acacia extract is catechin, epicatechin, or a combinationthereof.
 10. The composition according to claim 1, wherein thecomposition additionally comprises a glucosamine-type compound.
 11. Thecomposition according to claim 10 wherein the glucosamine-type compoundis selected from the group consisting of glucosamine sulfate,glucosamine hydrochloride, N-acetylglucosamine, chondroitin sulfatemethylsulfonylmethane, and hyaluronic acid.
 12. The compositionaccording to claim 1, wherein the composition further comprises apharmaceutically or nutraceutically acceptable carrier, diluent, orexcipient, wherein the pharmaceutical or nutraceutical formulationcomprises from about 0.5 weight percent (wt %) to about 90 wt % ofactive ingredients of the extract mixture.
 13. The composition accordingto claim 12, wherein the composition is formulated as a tablet, hardcapsule, softgel capsule, powder, or granule. 14.-30. (canceled)
 31. Amethod for (a) increasing bone density, (b) supporting or managing bonehealth, cartilage health or both, (c) supporting a normal and healthyrange of bone density, (d) reducing the action of harmful enzymes thatbreak down bone tissue, cartilage tissue or both, (e) altering theaction of enzymes that affect bone health, cartilage health or both, (f)improving bone structure, cartilage structure or both, or maintainingbone function, cartilage function or both, (g) improving physicalmobility, (h) alleviating bone pain, cartilage pain, stiffness or anycombination thereof, (i) managing and maintaining physical mobility, (j)improving range of motion and/or flexibility, (k) improving physicalfunction of bone, cartilage or both, (l) promoting or enhancingflexibility and comfortable movement, (m) supporting bone health,cartilage health or both, (n) promoting healthy bone function, cartilagefunction, bone comfort, cartilage comfort, or any combination thereof,(o) relieving bone discomfort, cartilage discomfort or both, (p)promoting healthy bone, cartilage or both, (q) maintaining bone,cartilage or both, (r) supporting joint cartilage, (s) treating,preventing, or managing cartilage degradation, (t) minimizing cartilagedegradation, (u) promoting flexible joints and strong cartilage, (v)maintaining steady blood flow to bone to support enhanced bone strength,or (w), in a mammal comprising administering an effective amount of acomposition according to claim
 1. 32.-45. (canceled)
 46. A method forrelieving bone discomfort, cartilage discomfort or both in a mammalcaused by exercise, work, overexertion or any combination thereof,comprising administering an effective amount of a composition accordingto claim
 1. 47.-51. (canceled)
 52. A method for promoting bone health,cartilage health, bone comfort, cartilage comfort, or any combinationthereof by maintaining synovial fluid for joint lubrication in a mammal,comprising administering an effective amount of a composition accordingto claim
 1. 53.-56. (canceled)
 57. A method for promoting bone comfortand a wide range of motion in a mammal after exercise, work,overexertion or any combination thereof, comprising administering aneffective amount of a composition according to claim
 1. 58. A method fortreating bone disorder, cartilage disorder or both in a mammal,comprising administering an effective amount of a composition accordingto claim
 1. 59. The method according to claim 58, wherein the bone orcartilage disorder is osteoporosis, osteoarthritis, osteonecrosis,osteophyte, bone fracture, metabolic bone disorders, osteochondritisdiseases, osteochondroma, osteitis deformans, osteitis fibrosa cystica,ostteitis pubis, condensing osteitis, osteogenesis imperfecta,osteomalacia (rickets), osteomyelitis, osteopenia, or any other bone andcartilage associated indication or any combination thereof. 60.-62.(canceled)
 63. The method according to claim 58, wherein an effectiveamount of the composition promotes the reduction the levels of CTX-II.